Proliferated cell lines and uses thereof

ABSTRACT

The subject invention pertains to tumor cell lines useful for increasing the proliferation potential of any human or animal cell in culture, thereby providing immortalized or continuous cell lines and cultures. The invention also concerns proliferation factors, and compositions containing the factors, which are capable of increasing the proliferation potential of any human or other animal cell in culture. The subject invention further pertains to a method for proliferation cells in culture by contacting cells with the proliferation factors. The proliferated cells can range in plasticity and can include, for example, blast cells, fertilized ova, non-fertilized gametes, embryonic stem cells, adult stem cells, precursor or progenitor cells, and highly specialized cells. Optionally, the cells can be induced to cease proliferation. The proliferation cells of the subject invention are useful for cell therapy, cell/gene therapy, biological production of molecules, and as in vitro models for research, toxicity testing, and drug development.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of priority of U.S.Provisional Application Ser. No. 60/355,157, filed Feb. 8, 2002, whichis hereby incorporated by reference herein in its entirety, includingany figures, tables, nucleic acid sequences, amino acid sequences, ordrawings.

BACKGROUND OF THE INVENTION

Most cells can be cultured in vitro to a limited extent usingconventional cell culture technology, provided that suitable nutrientsand other conditions for growth are supplied. Such cultures have beenused to study genetic, physiological, and other phenomena, as well as tomanufacture certain biomolecules using various fermentation techniques.In studies of mammalian cell biology, cell cultures derived from lymphnodes, muscle, connective tissue, kidney, dermis, and other tissuesources have been used, for example. However, most normal cells have alimited growth potential in culture. After a certain number of celldivisions (the Hayflick limit), they can no longer proliferate (HayflickL., Exp. Cell. Res., 1965, 37:614–636). This limited life span, termedreplicative senescence, likely arose as a protective mechanism againstunfettered clonal evolution and cancer in long-lived animals. Therefore,while it has long been a goal of scientists to be able to maintain alltypes of cells in vitro, standard culture conditions do not promote thelong-term survival or proliferation of most cells.

“Immortalization” is the escape from the normal limitation on growth ofa finite number of division cycles. Therefore, once immortalized, a cellline can be continuously cultured. However, immortal cell lines veryrarely emerge spontaneously under usual culture conditions.

In order to increase the life span of cells in culture, publishedtechniques have included the use of embryonic cells. The strategy ofstarting with embryonic cells is based on the fact that embryonic cellsare relatively less differentiated than adult cells, and thus can beexpected to go through several cycles of cell division before becomingterminally differentiated. It is an axiom of biology thatundifferentiated cells proliferate at a greater rate than differentiatedcells. It is generally believed that by the time a cell has developedthe necessary intra-cellular machinery for hormone synthesis andsecretion, for example, it is no longer able to divide rapidly, if atall.

Another known strategy for establishing cells in culture is to startwith tumor cells, due to their greater potential for proliferation.While these types of cell lines are able to generate a large number ofcells, the limited number of these types of lines, the limited number ofphenotypes that they are able to generate, and their inherenttumorgenicity, make these types of cell lines less than ideal.

Normal cells have been transformed in culture by various means includingthe use of UV light, chemical carcinogens, and the introduction ofoncogenes, which alters the genetic programming of the cell, therebyinducing the cell to proliferate indefinitely. Simian virus 40 (SV40)has been used for some time to immortalize human cells from differenttissues in order to gain continuously growing cell lines (Sack, G. H. Invitro, 1981, 17:1–19). Rat granulose cells were transformed byco-transfection with the entire SV40 genome and the activated Ha-rasgene (Baum, G. et al. Develop Biol, 1990, 112:115–128). These cells werereported to retain at least some differentiated characteristics, i.e.,they were able to synthesize steroids in response to cAMP. It has alsobeen shown that expression of SV40 large T protein alone is sufficientto induce transformed properties in primary cells (Abcouver, S.Bio/Technology, 1989, 7:939–946). Other cell lines established inculture include UMR cells, derived from normal islets of neonatal rats(NG, K. W. et al., J. Endocrinol., 1987, 113:8–10) and HIT cells,derived by SV40 infection of hamster islets (Santerre, R. F. et al.,PNAS, 1981, 78:4339–4343). The insulin secretory output of these celllines is low, however, and response to glucose is lost with passage inculture. Thus, while the proliferative status of these cell lines mayprove useful for studying the decisions that occur during celldetermination and differentiation, and for testing the effects ofexogenous agents, these immortalization agents may affect otherproperties of the cell, such as the cell's ability to differentiate andexpress genes in a physiologically correct manner.

More recent methods of cell line immortalization that are still in thebeginning stages of development involve telomeres, the ends ofchromosomes composed of non-coding repeat DNA sequences. It has beensuggested that the limited reproductive lifespan of normal (diploid)cells in culture may be explained by an inevitable shortening of one ormore telomeres. It is known that cancer cells, germ cells, and someeucaryotic microorganisms have the ability to correct this phenomenonwith the enzyme telomerase, which catalyzes telomere elongation. Normalcells modified to express telomerase are immortal in culture (Bodnar etal., Science, 1998, 279(5349):349–352), presumably by maintaining aconstant telomere length. Furthermore, in vitro-aged fibroblasts treatedwith telomerase regain dermal function (Funk et al., Exp. Cell. Res.,2000, 258(2):270–278).

Only a few neuronal cell types have been reported to divide in the adultbrain and adult neurons do not survive well in vitro. The generation ofclonal cell lines from different regions of the brain would greatlyfacilitate the discovery of new neurotrophic factors and theirreceptors, and enhance the understanding of their function. The centralnervous system contains two major classes of cells known as neurons andglial cells. There are hundreds of different types of neurons and manydifferent neurotrophic factors that influence their growth anddifferentiation. Depending upon the type of neuron and the region of thebrain in which the neuron resides, a different neurotrophic factor orspecific combination of factors affect the survival, proliferation, anddifferentiation of the neuron.

To date, neuropharmacological studies in the central nervous system(CNS) have been delayed by the lack of cell systems needed toinvestigate potentially useful neuroactive compounds. In live animals,the complexity of the brain makes it difficult to effectively measurewhich cellular receptors are being targeted by these compounds.Additionally, the expense involved in live animal research and thecurrent controversies stemming from animal rights movements have made invivo animal studies less acceptable for initial research. Primary cellsfrom neuronal tissue are often used for CNS studies; however, long-termculture of primary neurons has not been achieved. Only a few attempts toachieve long term culture and proliferation of neuronal cells have beenreported. In fact, the proliferation of neuronal cells has proven soelusive that it has become ingrained in the scientific community thatneuronal cells do not proliferate in vitro. As a consequence, freshdissections must be performed for each study in order to obtain thenecessary neuronal cell types, resulting in costly research withincreased variability in the experimental results.

While some neuronal tumor cell lines exist, they are few in number andare not well characterized. In general, these tumor cell lines do notmimic the biology of the primary neurons from which they were originallyestablished. In vitro primary cultures that are more phenotypicallyrepresentative of primary cells and that could generate continuouscultures of specific neuronal cell lines capable of proliferation wouldbe invaluable.

Similar to neurons, the endocrine cells of the mammalian pancreas havebeen considered to be post-mitotic, i.e., terminal, essentiallynon-dividing cells. Recent work has shown that the cells of themammalian pancreas (including those of humans) are capable of survivalin culture, but are not capable of sustained cell division. Hence, aprimary culture of the tissue cells can succeed, but due to a lack ofsufficient cell divisions of the cultured cells, passaging of theprimary culture to form serial cultures has not been possible. Althoughoccasional cells in a metaphase stage, uptake of tritiated thymidine,and other evidence of cell division have been seen in these cultures(Clark et al., Endocrinology, 1990, 126:1895; Brelijie et al.,Endocrinology, 1991, 128:45), the overall rate of cell division has beenconsidered to be below the replacement rate (that is, more, or as many,cells die as are produced).

The culture of animal cells in vitro, as “biofactories,” for theproduction of various proteins, peptides, hormones, growth factors, andother biologically active substances has been widely investigated. Forexample, pituitary cells have been cultured in vitro to produce growthhormone; kidney cells have been cultured to produce plasminogenactivator; and hepatitis-A antigen has been produced in cultured livercells. Other cells have been specifically cultured to produce variousviral vaccines and antibodies. Interferon, insulin, angiogenic factor,fibronectin and numerous other biomolecules have been produced by the invitro culture of various animal cells. Of course, the quantity ofbiomolecules produced by these biological factories is limited by thenumbers of cells and range of cell types available.

Various cell lines have also been used in animal models oftransplantation for a variety of purposes. Fetal kidney cells andamniotic cells have been transplanted as sources of trophic factors.Adrenal medullary cells, sympathetic ganglion cells, and carotid bodycells have been transplanted as sources of dopamine. Fibroblasts andglial cells have been transplanted as sources of trophic factors, tocarry genes through recombinant strategies, or for demyelinatingdiseases, for example. Corneal endothelial cells have been used forcorneal transplants. Myoblasts have been transplanted for the treatmentof muscular dystrophy and cardiac disease. Other cell lines includepancreatic islet cells for diabetes; thyroid cells for thyroiddisorders; blood cells for AIDS, bone marrow transplant, and inheriteddisorders; bone and cartilage for osteoarthritis, rheumatoid arthritis,or for fracture repair; skin or fat cells for reconstructive purposes,such as in skin grafts after burns or cosmetic surgery; breastaugmentation with fat; hair follicle replacement; liver cells for liverdisorders inducing hepatitis; and retinal pigment epithelial cells (RPE)for retinitis pigmentosa and Parkinson's disease.

Unfortunately, the inability to procure large numbers of primary cellsthat are genetically stable has impeded the ability of medical scienceto progress in the area of cell transplant therapy. In addition, currentsources for therapeutic donor cells are limited further by the inherentbiological variability among the donors.

Stem cells are believed to have immense potential for therapeuticpurposes for numerous diseases. Stem cells have been derived fromnumerous donor sources, including, but not limited to, embryonic, blast,tissue-derived, blood, and cord-blood cells; organ-derived progenitorcells; and bone marrow stromal cells; among others. Such stem cells canbe differentiated along numerous pathways to produce virtually any celltype. These cells can be transplanted either before or afterdifferentiation. From a therapeutic perspective alone, such cells may beuseful for the treatment of a vast array of disorders. Examples ofneurological disorders that can potentially be treated with stem cellsinclude Parkinson's disease, Alzheimer's and Huntington's diseases, ALS,stroke, demyelinating disorders, epilepsy, head trauma, and spinal cordinjury. However, stem cells share the same problem with other cellsrelating to the ability to proliferate the cells in vitro in sufficientquantities for diagnostic, investigational, or therapeutic purposes.Moreover, primary stem cells that have exhibited the most plasticity areembryonic stem cells. Obtaining large quantities of these cells isparticularly problematic and raises ethical issues.

The above description of the state-of-the-art makes it apparent thatthere is a need for methods to maintain any and all cells in long-termcultures at increased proliferation rates, thereby providing a moreplentiful and less costly supply of cells. Such long-term cultures couldbe developed as biological “factories” for the production oftherapeutically useful proteins, for example. Well-established celllines would also offer the possibility of in vitro bioassays based onthe cells' responses to drugs and other chemicals (e.g., for toxicityand efficacy studies). There is also a need for the ability to produce ahomogenous cell line, particularly a homogenous cell line of humanorigin. The availability of cells and cell lines that can becryo-preserved is likewise lacking.

Continuously cultured cell lines would also be invaluable as a source ofcells for cell transplant therapy, which has been found effective incorrecting many disease states. For instance, diabetics could bestabilized and possibly cured through the implantation of cells thatreplace the function of insulin-secreting β-cells of the pancreas.Parkinson's patients could be treated with a ready supply ofdopaminergic neurons, or stem cells giving rise to dopaminergic neurons.Such cell lines would also provide an endless supply of cells and tissuereadily accessible for genetic modulation in vitro prior to transplant,for use in cell-mediated gene therapy. Thus, there exists a need formethods to produce cells and cell lines that would proliferate forextended periods in vitro yet faithfully retain their differentiatedfunctions.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to tumor cell lines useful for increasingthe proliferation potential of cells, including cultures of human andother animal cells. The subject invention particularly pertains to a ratthyroid cell line (UCHT1) useful for increasing the proliferationpotential of cells. The subject invention also concerns conditionedmedium prepared from such tumor cell lines, and other tumor cell lineextracts. The conditioned medium of the invention can be used to produceimmortalized or continuous cell lines. The invention further pertains tothe cell lines immortalized using the conditioned medium of the subjectinvention.

In a further aspect, the subject invention concerns a proliferationfactor obtainable from a tumor cell line, such as the UCHT1 cell line,as well as methods of using a tumor cell line, its proliferation factor,tumor cell line conditioned medium, and/or other tumor cell lineextracts, to increase the proliferation potential of cells. Theproliferation factor is obtainable from tumor cells lines of variousspecies, particularly mammalian species, such as rats and humans. Thesubject invention also concerns cell lines immortalized using a tumorcell line proliferation factor, or using compositions (e.g., conditionedmedium and/or other tumor cell line extracts) containing such tumor cellline proliferation factors. In a specific embodiment, the proliferationfactor is from about 30 kD to about 100 kD. The subject inventionfurther pertains to fragments, analogues, or derivatives of thefull-length tumor cell line proliferation factor. The methods of thesubject invention can be used to enhance the proliferation potential ofcells, including proliferation duration and/or proliferation rate. Forexample, the methods of the subject invention produce cell lines thatproliferate indefinitely, and intervals between consecutive divisions ofa cell as little as 24 hours can be achieved. Further, the cells of thesubject invention can be grown in large-scale culture and cryopreservedwith full retention of viability in vitro and in vivo.

In another aspect, the subject invention concerns methods fortransplanting cells to a patient in need thereof. These methods can beused for alleviating the symptoms of a variety of disorders or trauma byadministering proliferated cells of the invention to a patient (e.g., ahuman or other animal) in need thereof. For example, proliferated cellsof the subject invention can be administered to a patient suffering froma pathological condition, such as a condition associated with celldeath, cell loss, or cell dysfunction. Advantageously, using the methodsof the subject invention, immortality can be conferred to cell lineswithout the necessity for incorporation of an oncogene. Therefore, themajority of proliferated cell lines produced by the methods of thesubject invention are non-tumorgenic in vivo.

The proliferated cells of the invention can range in plasticity fromtotipotent or pluripotent stem cells (e.g., adult or embryonic),precursor or progenitor cells, to highly specialized cells, such asthose of the central nervous system (e.g., neurons and glia). Theproliferated stem cells of the subject invention can be obtained from avariety of sources, including embryonic tissue, fetal tissue, adulttissue, umbilical cord blood, peripheral blood, bone marrow, and brain,for example. Blast cells can be proliferated using the methods of thesubject invention.

Using methods of the subject invention, stem cells can be modified, thensubsequently proliferated. For example, stem cells can be modifiedthrough genetic modification (e.g., genetic engineering), differentiatedwith differentiation agents (e.g., trophic factors), or with adjuvants(e.g., chemotherapies, radiation therapies, and the like), thenproliferated. Alternatively, stem cells can be proliferated, thensubsequently modified.

Using methods of the subject invention, non-stem cells (e.g.,specialized or mature cells, such as dopamine-producing neurons, ortheir precursors or progenitors) can be modified, then subsequentlyproliferated. For example, non-stem cells can be modified throughgenetic modification (e.g., genetic engineering), differentiated withdifferentiation agents (e.g., trophic factors), or with adjuvants (e.g.,chemotherapies, radiation therapies, and the like), then proliferated.Alternatively, non-stem cells can be proliferated, then subsequentlymodified.

Cells of the subject invention, including B-cells and T-cells, forexample, can be genetically modified to produce various biomolecules,such as trophic factors or antibodies, as well as to exhibit any numberof bioactive properties. Cells can be genetically modified before,during, or after proliferation with a tumor cell line proliferationfactor of the invention.

As will be understood by one of skill in the art, there are over 200cell types in the human body. It is believed that the methods of thesubject invention can be used to proliferate any of these cell types fortherapeutic or other purposes. For example, any cell arising from theectoderm, mesoderm, or endoderm germ cell layers can be proliferatedusing methods of the subject invention. It will be understood by one ofskill in the art that the methods of the present invention are alsoapplicable for veterinary purposes. For example, cells of non-humananimals can find application either in human or animal patients (e.g.,veterinary uses). Although dopamine neurons from human, pig, and rat aresimilar in that they synthesize dopamine and release synaptically intothe brain, they differ immunologically, in extent of reinervation of thebrain, in life span, and in infection agents associated with thespecific donor or donor species. These traits can be exploited for theirspecific strengths and weaknesses.

The subject invention provides a ready source of cells for research,including pharmacological studies for the screening of various agents,and toxicologic studies for the cosmetic and pharmaceutical industries.The subject invention further provides cells that can be used asbiofactories, for the large-scale production of biomolecules, eithernaturally or recombinantly.

The subject invention further pertains to nucleotide sequences, such asDNA sequences, encoding the proliferation factor of the subjectinvention disclosed herein, and the proliferation factor receptor. Thesenucleotide sequences can be synthesized by a person skilled in the art.The sequences may be used to genetically modify an appropriate host toconfer upon that host the ability to produce the proliferation factor orits receptor. Hosts of particular interest include vertebrate cellsdisclosed herein, bacteria, and yeast, for example. The subjectinvention also concerns vectors containing nucleotide sequences encodingthe proliferation factor or the proliferation factor receptor disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a method of the subject invention, wherein mediaconditioned by the rat thyroid UCHT1 cell line for 48 hours isfreeze-thawed 3 times in the absence of cryopreservants. The media isfiltered through 0.2 μm filters to yield a cell free conditioned media.Primary cultures of mammalian origin are kept in the presence of 10–20%(v/v) for the time range indicated. Successful immortalization isassessed by the generation of transformation foci in the culture.

FIGS. 2A–2F show phase contrast microscopic images of differentiated andundifferentiated RCSN cells. FIG. 2A (control) shows thatundifferentiated RCSN cells tend to exhibit an epithelial-likemorphology, with short or no processes and a more acidophylic cytoplasm.FIG. 2B shows that, after differentiation, cell proliferation is greatlyreduced, and RCSN cells develop processes and establish contact withneighboring cells. FIGS. 2C and 2D show hematoxilin and eosin (H-E)staining, before and after differentiation, respectively. FIGS. 2E and2F show melanin staining, before and after differentiation,respectively, using the ferrous ion capture technique, demonstrating ahomogenous distribution of the pigment in cytoplasm, with faint labelingin undifferentiated stages and a substantial increase upondifferentiation.

FIGS. 3A–3H show immunohistochemistry for neuronal markers. FIGS. 3A and3B show RCSN cells stained for neuron specific enolase (NSE), before andafter differentiation, respectively. FIGS. 3C and 3D show RCSN cellsstained for synaptophysin (SNP), before and after differentiation,respectively. FIGS. 3E and 3F show RCSN cells stained for microtubularassociated protein-2 (MAP-2), before and after differentiationrespectively. FIGS. 3G and 3H show images of differentiated RCSN cellstaken under epifluorescence conditions and stained for neurofilament andtetanus toxin, respectively.

FIGS. 4A–4D show immunohistochemical staining and micrographs of RCSNcells. FIGS. 4A and 4B show immunohystochemical staining for tyrosinehydroxilase (TH), under undifferentiated (control) and differentiatedconditions, respectively. FIGS. 4C and 4D show micrographs using theferrous ion capture technique, where fluorescent areas representcatecholamine deposits.

FIG. 5 shows Ca²⁺ in fluo-3 loaded RCSN lines. The image shows cellsthree seconds after being stimulated with the addition of 200 μmglutamate, and even more intensely when using simultaneous depolarizingconditions (70 mM K⁺). Fluorescence intensity is depicted in a pseudocolor scale, which in ascending order isblack-blue-green-yellow-orange-red.

FIGS. 6A and 6B show graphs representing patterns of decrease in therate of rotation after transplant of RCSN-3 cells into the striatum of 6hydroxy dopamine (6 OHDA)-lesioned rats.

FIGS. 7A–7C show micrographs of striatal sections of two rats sacrificed16 weeks after RCSN-3 cell transplantation. FIGS. 7A and 7C show RCSN-3cells immunostained with tyrosine hydroxilase (TH) at 10× and 40×magnification, respectively. FIG. 7B shows RCSN-3 cells immunostainedfor DOPA decarboxilase (DOPA-DC) at 40× magnification.

FIG. 8 shows a panoramic view of a brain section of a control ratwithout injury (lesion) or transplant of RCSN-3 cells. Strong browncolored zones correspond to TH-positive (TH⁺) cells. The normal ratdisplays symmetry in the labeling of both hemispheres, where thestriatum (top arrow) and the substantia nigra (SN) (lower arrow) exhibitTH.

FIG. 9 shows a panoramic view of a brain section of a control rat with a6 hydroxy dopamine (6-OHDA)-induced lesion of the ventral tegmentalarea, without transplant of RCSN-3 cells. A marked difference inlabeling is observed in the region of the striatum (black arrow). Thisfigure confirms the destruction of dopaminergic terminals in the rightstriatum that proceeded from the nigrostriatal pathway.

FIG. 10 shows a panoramic view of a section of the rat brain in theexperimental group (i.e., 6-OHDA-induced lesion, and transplanted withRCSN-3 cells). A TH⁺ zone (arrow, circle) corresponds to an accumulationof transplanted RCSN-3 cells, near the lateral ventricle (circle).

FIG. 11 shows a photomicrograph of the implant zone within the brain ofa lesioned rat transplanted with RCSN-3 cells. TH⁺ reaction is observed.Arrows indicate the accumulation of TH⁺ cells surrounding the area ofthe needle tract (40× magnification). The neuronal density of implantsdoes not permit distinction of individual neurons clearly.

FIG. 12 shows a photomicrograph of implanted RCSN-3 cells with neuronalmorphology in the striatum of a lesioned recipient rat (100×magnification). Note the presence of process (thicker, upper arrows)extending from the somas (thin, lower arrows).

FIG. 13 shows a photomicrograph of implanted RCSN-3 cells in thestriatum of a lesioned recipient rat (100× magnification). Note theexistence of a significant number of processes oriented predominantlytoward the striatum.

FIG. 14 shows a photomicrograph of implanted RCSN-3 cells within thestriatum of a lesioned recipient rat (100× magnification). The presenceof TH⁺ somas single lower grey arrow and processes (four upper blackarrows).

FIGS. 15A–15E show lower magnification photomicrographs of rat striatum.FIGS. 15A–15C show sections at the striatum level (2×, 5×, and 5×magnification, respectively) of a lesioned rat brain transplanted withRCSN-3 cells, with a TH⁺ area in the middle of each section. In FIG.15C, the diaphragm of the microscope is closed to contrast striasomes,and the TH⁺ surrounds them (right) and in a linear projection (left).Somas are not evident at this magnification. FIGS. 15D and 15E showlesioned controls of rat striatum.

FIGS. 16A–16C show amperimetric detection of dopamine. FIG. 16A showscalibration using 25 μM dopamine. FIGS. 16B and 16C show amperimetricsignals of dopamine in RCSN-3 cells, after depolarizing stimulation with70 mM external K⁺. Deflections corresponding to dopamine are present,demonstrating that RCSN cells are capable of production and activesecretion of dopamine in vitro.

FIGS. 17A–17D show controls for the evaluation of1-methyl-4-phenylpyridinium (MPP⁺) production by cell lysates incubatedwith 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). FIG. 17A showstissue culture media (control). FIG. 17B shows phosphate buffered saline(PBS) with antiproteases (AP) (control). FIG. 17C shows 10 μM MPTP withPBS and AP (control). FIG. 17D shows 10 μM MPP⁺ with PBS and AP(control).

FIGS. 18A–18C show the evaluation of MPP⁺ production by RCSN-3 cell(differentiated and non-differentiated) lysates incubated with MPTP.FIG. 18A shows 10 μM MPP⁺ in RCSN-3 cell extract. FIG. 18B shows 10 μMMPTP in RCSN-3 cell extract. FIG. 18C shows 10 μM MPTP in RCSN-3 cellextract from differentiated cells. As expected, no MPP⁺ peak is observedafter incubation with MPTP, suggesting the lack of an MAO B activity inRCSN cells.

FIGS. 19A and 19B show DNA fragmentation in the RCSN-3 cell line treatedwith MPP⁺ using the terminal deoxynucleotidyl transferase-mediated dUTPnick-end labeling technique (TUNEL). FIG. 19A shows RCSN-3 cells in theabsence of MPP⁺ (control) and FIG. 19B shows RCSN-3 cells treated withMPP⁺.

FIGS. 20A–20C show mitochondrial membrane potentials of RCSN-3 cells asdetected with potassium tetrachloride ortetraethylbenzimidazolilcarbocianine (JC-1). FIG. 20A shows untreatedRCSN-3 cells (control). FIG. 20B shows RCSN-3 cells in the presence ofdopamine. FIG. 20C shows RCSN-3 cells in the presence of manganese.

FIGS. 21A and 21B show mitochondrial membrane potentials of RCSN-3cells. FIG. 21A shows untreated RCSN-3 cells (control). FIG. 21B showsRCSN-3 cells in the presence of MPP⁺.

FIGS. 22A and 22B show the ratio of emission of JC-1 monomer versusemission of “J” aggregate (Em(520 nm)/Em(590 nm)) in RCSN-3 cells.

FIG. 23 shows the results of melanin experiments conducted on RCSN-3cells in the presence or absence of levodopa (L-Dopa). FIG. 23 showsexperimental results using four experimental conditions: (i) plasticdishes in the absence of L-Dopa; (ii) plastic dishes in the presence ofL-Dopa; (iii) glass dishes in the absence of L-Dopa; and (iv) glassdishes in the presence of L-Dopa.

FIG. 24 shows SDS-PAGE demonstrating expression of brain-derivedneurotrophic factor (BDNF) and the glucocorticoid receptor (GR) in cellsof the H1b cell line (normal fetal mouse hippocampal cells with neuronalphenotype), which were previously cultured in UCHT1 conditioned medium,and were either proliferating (P) or differentiated (D).

FIG. 25 shows SDS-PAGE dyed with Coomassie blue stain at everysaturation level in precipitation with ammonium sulfate, as shown inTable 5. The first column contains molecular weight markers. FIG. 25shows that the main components are located at approximately 65 kD and 15kD, which are associated with albumin and lactoalbumin, respectively.Most protein precipitates at 65%–80% of saturation with ammoniumsulfate, but there are a greater number of proteins in the range of40%–50% and 50%–65%. Theoretically, thyroglobulin precipitates at40%–50%.

FIG. 26 shows curves associated with samples in ionic exchangechromatography. Although scales are not comparable, the sensitivity ofthe method is determined by the major components. The curve of serum andculture media may have slight differences due to the presence of aminoacids and salts in the basal media. Greater differences betweenconditioned media and culture media would suggest the presence ofsecreted proteins. However, after correcting for actual protein content,no significant differences were found to justify a direct comparativeanalysis.

FIG. 27 shows anionic exchange chromatography (DEAE cellulose). Buffersolutions are Bis-Tris 20 mM pH 7 for balance and binding, and Bis-Tris20 mM pH 7 1M NaCl for elution, considering 20 volumes of column. Whencompared to FIG. 26, the patterns for transferrin and bovine serumalbumin (BSA) are clearly identifiable in fractions 10 and 13–22,respectively. A break exists in the peak for BSA, related to themaintenance of a 20% gradient of the molarity of the salt.

FIG. 28 shows anionic exchange chromatography (DEAE cellulose). Theresolution between pure albumin and transferrin is 1.7, which is lessthan that seen for conditioned media (as shown in FIG. 26), where aresolution of 0.89 for equivalent peaks can be seen. Ideally, resolutionlevels should be 1.5 or more.

FIG. 29 shows an isolectric focusing gel (IEF) of DEAE-cellulosechromatographic fractions of conditioned media. Albumin predominates inthe indicated fractions, but the effect is attenuated in the extremitiesof the peak, although not enough to allow an adequate resolution of theremaining proteins.

FIG. 30 shows hydrophobic interaction chromatography, presenting thevisible peak for the scale associated to the concentrations for the maincontaminants. These peaks are readily identifiable after comparing thechromatographic profile in a similar study done in with pure albumin andtransferrin (FIG. 31). Correspondence is not identical, possibly due tonon-specific hydrophobic interactions associated with the mixture ofmedia proteins. Resolution is superior to 2.

FIG. 31 shows hydrophobic interaction chromatography for pure albuminand transferrin. Resolution is superior to 1.9.

FIGS. 32A–32D show the results of bioassays using the KGFR cell line.HSS and conditioned media exert a proliferating effect, although nosignificant differences are apparent among them. The pro-proliferativeeffect is evident in FIG. 32B.

FIG. 33 shows a control SDS-PAGE dyed with Coomassie blue to determinepossible protein loss during the pretreatment of the conditioned media.The results show that the loss due to desalinization is negligible.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention pertains to tumor cell lines, such as the Fisher344 rat thyroid cell line (UCHT1), useful for increasing theproliferation potential of cell cultures, including cultures of humanand other animal cells, such that immortalized or continuous cellcultures are produced. The subject invention also concerns conditionedmedium prepared from such tumor cell lines, which can also be used toproduce immortalized or continuous cell lines. The invention furtherpertains to cell lines immortalized using conditioned medium of thesubject invention.

In a further aspect, the subject invention concerns a proliferationfactor produced by tumor cell lines, and methods of using tumor celllines, their proliferation factor, conditioned medium, and/or othertumor cell line extracts, to increase the proliferation potential ofcells. The subject invention also concerns cell lines immortalized usinga tumor cell line proliferation factor, or using compositions (e.g.,conditioned medium and/or other tumor cell line extracts) containingsuch tumor cell line proliferation factors. Conditioned medium caninclude medium in which the tumor cell lines of the subject invention(e.g., UCHT1) have been grown, wherein the proliferation factor issecreted or is otherwise delivered to the medium by the tumor cell line.In a specific embodiment, the proliferation factor is from about 30 kDto about 100 kD. The subject invention further pertains to polypeptidesrepresenting fragments, analogues, or derivatives of the full-lengthtumor cell line proliferation factor, or fusion proteins comprising suchsequences, wherein the polypeptides retain some or all of thecharacteristic proliferating activity of the tumor cell lineproliferation factors disclosed herein. The methods of the subjectinvention can be used to produce continuous cell lines that proliferateindefinitely. Many of these propagated cell lines have been maintainedin vitro for one year and longer, and some for over 10 years, withretention of their differentiation markers. Using the methods of thepresent invention, it is possible to achieve a cell division period aslittle as 24 hours, or less.

The cells of the subject invention can be grown in large-scale cultureand cryopreserved with substantial retention of viability.Advantageously, using the methods of the subject invention, cells can betransformed and continuous cell lines can be created without thenecessity for incorporation of an oncogene within the target cell line.Hence, a tumor cell line, its proliferation factor, conditioned mediumor other extract obtained from a tumor cell line, permits enhancedand/or sustained proliferation of a target cell. The tumor cell line canbe derived from a wide variety of mammal species, including human. Inone embodiment, the tumor cell line is a thyroid tumor cell line. Inanother embodiment, the tumor cell line is a rodent thyroid tumor cellline (e.g., rat or mouse cell line). In a still further embodiment, thetumor cell line is a rat thyroid cell line. In a specific embodiment,the tumor cell line is the rat thyroid tumor cell line, UCHT1.

The method for proliferating cells according to the subject invention,thereby producing immortalized or continuous cell lines, comprises thestep of contacting a target cell or cells with a tumor cell lineproliferation factor, such as the UCHT1 cell line proliferation factor.The proliferation factor induces or promotes the proliferation of thecells. In one embodiment, the method comprises culturing target cells inprimary culture with conditioned medium from a tumor cell line, such asthe Fisher 344 rat thyroid cell line, UCHT1. After a period of time inthe range of about 1–8 months, cells become transformed into acontinuously dividing but differentiated state. However, it should beunderstood that the duration of exposure to (e.g., contact with) a tumorcell line proliferation factor necessary to produce the continuous celllines of the subject invention can vary with the type of target cell andthe conditions under which contact is made. For example, durations ofexposure shorter than one month and longer than eight months are alsocontemplated. The method for proliferating cells can also include thestep of isolating the cell or cells from a human or other animal. Themethod for proliferating cells can optionally include a step of inducingthe cells to differentiate.

In another aspect, the invention concerns a composition forproliferating cells. The composition of the invention comprises aproliferation factor produced by a tumor cell line, such as the UCHT1cell line. In one embodiment, the composition is conditioned medium of atumor line, wherein the conditioned medium contains a tumor cell lineproliferation factor.

The tumor cell lines and proliferation factors of the subject inventionare not the teratocarcinoma stem cell line (PSA-1) or factor describedin Martin G. R., Proc. Natl. Acad. Sci. USA, December 1981,78(12):7634–7638.

Various culturing methods known in the art can be used to contact thetarget cells with a tumor cell line proliferation factor (orcompositions containing a proliferation factor) for a period of time,and in such a way that target cells are transformed and continuouscultures are produced. Propagation can be carried out under in vitroconditions, such as in suspension cultures or by allowing cells toadhere to a fixed substrate, or under in vivo conditions. For example,using a container with large growth surfaces, such as round bottles,cells can be grown in a confluent monolayer. The bottles can be rotatedor agitated in motorized devices to keep the cells in suspension (e.g.,the “roller flask” technique). Roller culture apparatus and similardevices are commercially available (WHEATON SCIENCE PRODUCTS).

The cells of the subject invention can be proliferated in culture asheterogeneous mixtures of cells or cell types, or clonally. A cell issaid to be clonally derived or to exhibit clonality if it was generatedby the division of a single cell and is genetically identical to thatcell. Purified populations (clonal lines) are particularly useful for invitro cell response studies, efficient production of specificbiomolecules, and cell transplant therapy, because the exact identity ofthe cells' genetic capabilities and functional qualities are readilyidentified.

In order to produce the continuous cell lines of the subject invention,the target cells can be exposed to the tumor cell line proliferationfactors disclosed herein by various methods known in the art.Furthermore, various techniques of isolating, culturing, andcharacterizing cells can be utilized to carryout the method of thesubject invention, including those techniques described in Freshney R.I., ed., (2000), Culture of Animal Cells: A Manual of Basic Technique,Fourth edition, Wiley-Liss, New York. For example, the target cells canbe exposed to a tumor cell line proliferation factor in the presence, orabsence, of various substances, such as serum or other trophic factors.

A wide variety of media, salts, media supplements, and products formedia formulation can be utilized to produce the continuous cell linesof the subject invention, depending upon the particular type of targetcell. Examples of these substances include, but are not limited to,carrier and transport proteins (e.g., albumin), biological detergents(e.g., to protect cells from shear forces and mechanical injury),biological buffers, growth factors, hormones, hydrosylates, lipids(e.g., cholesterol), lipid carriers, essential and non-essential aminoacids, vitamins, sera (e.g., bovine, equine, human, chicken, goat,porcine, rabbit, sheep), serum replacements, antibiotics, antimycotics,and attachment factors. These substances can be present in variousclassic and/or commercially available media, which can also be utilizedwith the subject invention. Examples of such media include, but are notlimited to, Ames' Medium, Basal Medium Eagle (BME), Click's Medium,Dulbecco's Modified Eagle's Medium (DMEM), DMEM/Nutrient Mixture F12Ham, Fischer's Medium, Minimum Essential Medium Eagle (MEM), NutrientMixtures (Ham's), Waymouth Medium, and William's Medium E.

The UCHT1 cell line was deposited with the following InternationalDepository Authority (IDA): Deutsche Sammlung Von Mikroorganismen undZellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig,Germany on Feb. 1,2002. The culture deposit number is DSM ACC2535.

The culture deposited for the purposes of this patent application wasdeposited under conditions that assure that access to the culture isavailable during the pendency of this patent application to onedetermined by the Commissioner of Patents and Trademarks entitledthereto under 37 C.F.R. § 1.14 and 35 U.S.C. § 122. The deposit will beavailable as required by foreign patent laws in countries whereincounterparts of the subject application, or its progeny, are filed.However, it should be understood that the availability of a deposit doesnot constitute a license to practice the subject invention in derogationof patent rights granted by government action.

Further, the subject culture deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe deposit of biological materials, i.e., they will be stored with allthe care necessary to keep them viable and uncontaminated for a periodof at least five years after the most recent request for the furnishingof a sample of the deposit, and in any case, for a period of at leastthirty (30) years after the date of deposit or for the enforceable lifeof any patent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of adeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

In another aspect, the subject invention pertains to nucleotidesequences, such as DNA sequences, encoding the tumor cell lineproliferation factor of the subject invention disclosed herein. Thenucleotide sequences include not only the native sequences but alsofragments of these sequences, analogues, and mutants of these sequences,wherein the encoded polypeptides retain some or all of thecharacteristic proliferating activity of the tumor cell lineproliferation factors disclosed herein. These nucleotide sequences canbe readily synthesized by a person skilled in the art. The sequences maybe used to genetically modify eukaryotic or prokaryotic cells, forexample, bacterial cells, mammalian cells, yeast cells or fungi cellsfor synthesis of the proliferation factor of the invention. Viruses mayalso be genetically modified using such polynucleotides, to serve asvectors for the delivery of the polynucleotides to host cells. Thus, inyet another aspect, the subject invention concerns vectors containingpolynucleotides encoding the tumor cell line proliferation factor of thesubject invention disclosed herein. Exemplary vectors include plasmids,cosmids, phages, viruses, liposomes, and lipid-conjugating carriers.

The various methods employed in the genetic modification of host cellsare well known in the art and are described, for example, in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, second edition,volumes 1–3, Cold Spring Harbor Laboratory, New York, and Gloves, D. M.(1985) DNA Cloning, Vol. I: A Practical Approach, IRL Press, Oxford.Thus, it is within the skill of those in the genetic engineering art toextract DNA from its source, perform restriction enzyme digestions,electrophorese DNA fragments, tail and anneal plasmid and insert DNA,ligate DNA, transform cells, e.g., prokaryotic and eukaryotic cells,prepare plasmid DNA, electrophorese proteins, and sequence DNA.

Using methods of the subject invention, stem cells can be modified, thensubsequently proliferated. For example, stem cells can be modifiedthrough genetic modification (e.g., genetic engineering), differentiatedwith differentiation agents (e.g., trophic factors), or with adjuvants(e.g., chemotherapies, radiation therapies, and the like), thenproliferated. Alternatively, stem cells can be proliferated, thensubsequently modified.

Using methods of the subject invention, non-stem cells (e.g.,specialized or mature cells, such as dopamine-producing neurons, ortheir precursors or progenitors) can be modified, then subsequentlyproliferated. For example, non-stem cells can be modified throughgenetic modification (e.g., genetic engineering), differentiated withdifferentiation agents (e.g., trophic factors), or with adjuvants (e.g.,chemotherapies, radiation therapies, and the like), then proliferated.Alternatively, non-stem cells can be proliferated, then subsequentlymodified.

Accordingly, stem cells and non-stem cells (e.g., specialized or maturecells, or their precursors or progenitors) can optionally be modifiedbefore, during, and/or after proliferation, using the methods of thesubject invention. The modification can be through one or more of thefollowing interventions: genetic modification, differentiation withdifferentiation agents, or with adjuvants, for example. Thedifferentiation induced can be partial differentiation or fulldifferentiation along any number of phenotypic pathways, and can includechanges to a cell's morphology and/or function.

Target Cells

The proliferated cells of the invention can be derived from humans orother mammals, including non-human primates, rodents, and pigs, forexample. Specific examples of source species include, but are notlimited to, apes, chimpanzees, orangutans, humans, monkeys; domesticatedanimals (pets) such as dogs, cats, guinea pigs, hamsters, Vietnamesepot-bellied pigs, rabbits, and ferrets; domesticated farm animals suchas cows, buffalo, bison, horses, donkey, swine, sheep, and goats; exoticanimals typically found in zoos, such as bear, lions, tigers, panthers,elephants, hippopotamus, rhinoceros, giraffes, antelopes, sloth,gazelles, zebras, wildebeests, prairie dogs, koala bears, kangaroo,opossums, raccoons, pandas, giant pandas, hyena, seals, sea lions,elephant seals, porpoises, dolphins, and whales. The target cells canalso be derived from non-mammals, such as fish.

The proliferated cells of the invention can range in plasticity fromtotipotent or pluripotent stem cells (e.g., adult or embryonic),precursor or progenitor cells, to highly specialized or mature cells,such as those of the central nervous system (e.g., neurons and glia).Stem cells can be obtained from a variety of sources, including fetaltissue, adult tissue, umbilical cord blood, peripheral blood, bonemarrow, and brain, for example. Stem cells and non-stem cells (e.g.,specialized or mature cells, and precursor or progenitor cells) can bedifferentiated and/or genetically modified before, during, or afterproliferation using the methods of the subject invention. As usedherein, the term “embryo” is intended to include the morula, blastocyst,gastrula, and neurula. For example, blast cells can be proliferatedusing the methods of the subject invention.

Cloned cells, fertilized ova, and non-fertilized gametes can also beproliferated according to the methods of the invention. For example,fertilized ova or non-fertilized gametes can be used for reproductivepurposes or cloning purposes.

Other cells that can be proliferated using methods of the subjectinvention include, but are not limited to, neural cells, includingnigral dopaminergic neurons of fetal, neonatal, and adult origins; glialcell lines from mesencephalon and striatum, of fetal, neonatal, andadult origins; GABAergic cells from various areas of the brain,including striatum or cortex, of fetal, neonatal, and adult origins;cholinergic neurons from the striatum, septum, and nucleus basalis offetal, neonatal, and adult origins; and serotogenic neurons derived fromthe lateral hypothalamus, dorsal raphe nucleus or hindbrain ofembryonic, neonatal, or adult origins. Glial cells from numerousregions, including mesencephalon, striatum, cortex, subcortical whitematter, spinal cord, or Schwann cells, of fetal, neonatal, and adultorigins.

As will be understood by one of skill in the art, there are over 200cell types in the human body. The methods of the subject invention areuseful in proliferating any of these cell types, for therapeutic orother purposes. For example, cells that can be proliferated using themethods of the subject invention include those cells arising from theectoderm, mesoderm, or endoderm germ cell layers. Such cells include,but are not limited to, neurons, glial cells (astrocytes andoligodendrocytes), muscle cells (e.g., cardiac, skeletal), chondrocytes,fibroblasts, melanocytes, Langerhans cells, keratinocytes, endothelialcells, epithelial cells, pigment cells (e.g., melanocytes, retinalpigment epithelial (RPE) cells, iris pigment epithelial (IPE) cells),hepatocytes, microvascular cells, pericytes (Rouget cells), blood cells(e.g., erythrocytes), cells of the immune system (e.g., B and Tlymphocytes, plasma cells, macrophages/monocytes, dendritic cells,neutrophils, eosinophils, mast cells), thyroid cells, parathyroid cells,pituitary cells, pancreatic cells (e.g., insulin-producing β cells,glucagon-producing α cells, somatostatin-producing δ cells, pancreaticpolypeptide-producing cells, pancreatic ductal cells), stromal cells,Sertoli cells, adipocytes, reticular cells, rod cells, and hair cells.Other examples of cell types that can be proliferated using the methodsof the subject invention include those disclosed by Spier R. E. et al.,eds., (2000) The Encyclopedia of Cell Technology, John Wiley & Sons,Inc., and Alberts B. et al., eds., (1994) Molecular Biology of the Cell,3^(rd) ed., Garland Publishing, Inc., e.g., pages 1188–1189.

Methods and markers commonly used to identify stem cells and tocharacterize differentiated cell types are described in the scientificliterature (e.g., Stem Cells: Scientific Progress and Future ResearchDirections, Appendix E1–E5, report prepared by the National Institutesof Health, June, 2001). The list of adult tissues reported to containstem cells is growing and includes bone marrow, peripheral blood,umbilical cord blood, brain, spinal cord, dental pulp, blood vessels,skeletal muscle, epithelia of the skin and digestive system, cornea,retina, liver, and pancreas.

According to methods of the subject invention, stem cells can be exposedto a tumor cell line proliferation factor by contact with the tumor cellline, tumor cell line conditioned medium, other tumor cell lineextracts, or by contact with the purified proliferation factor itself.The stem cells can be exposed to the tumor cell line proliferationfactor when the stem cells are at different stages of development, suchas the blast stage, progenitor stage, stem cell stage, as well ascommitted to differentiated progenitor stage. It would be expected thatthe dividing cells would maintain a differentiated state proportional tothe developmental stage in vitro, at which donor tissue is exposed tothe conditioned medium.

Using methods of the subject invention, stem cells can be modified withdifferentiation agents (e.g., trophic factors), through geneticmodification (e.g., genetic engineering), or with adjuvant (e.g.,chemotherapies, radiation therapies, and the like), then subsequentlyproliferated. Alternatively, stem cells can be proliferated, thensubsequently modified.

Undifferentiated stem cells can be cultured to a point where they arecommitted to becoming a particular cell type (e.g., dopamine neuron),then administered to a patient to complete their growth anddifferentiation within the host (e.g., within the host brain).Alternatively, less-committed stem cells can be administered to thepatient, relying on “environmental” signals to guide them into becomingthe appropriate type of replacement cells.

The cells of the subject invention can be induced to reduce theirproliferation rate to the point that proliferation is arrested. Forexample, if cells have been proliferated using the methods of thesubject invention such that their proliferation rate has been increasedfrom its basal rate in culture, proliferation can be induced to ceasesimply by removing the proliferating cells from contact with theproliferation factor, or removing the proliferation factor from contactwith the cells. If cells have been proliferated with the proliferationfactor for a period of time sufficient to immortalize the cells (thusproducing a continuous cell line) such that contact with theproliferation factor is no longer necessary to maintain proliferation,the cells can be induced to cease proliferation by differentiating thecells through differentiation protocols, such as serum deprivation, orcontacting the cells with one or more differentiation agents, asdescribed below. Advantageously, the cells of the subject invention canbe induced to cease proliferation prior to administration to a patient.

Although the methods of the subject invention permit the proliferationof cells with at least some retention of their differentiatedattributes, the cells of the subject invention can be induced todifferentiate further along particular developmental paths, dependingupon the particular cell's plasticity. For example, when cellproliferation is stopped, the cells of the subject invention can becategorized along a continuum that includes, but is not limited to,“wild type” cells having the exact cell type of the starting cellmaterial and “wild type-like” cells such that they retain at least someof the properties or produce at least of the products of the startingcells, but not having fully differentiated into the starting cell type.

Depending upon cell type, differentiation of the cells can be induced byany method known in the art that activates the cascade of biologicalevents that lead to cell growth. For example, cells can be induced todifferentiate by plating the cells on a fixed substrate, such as aflask, plate, or coverslip, or a support of collagen, fibronectin,laminin, or extracellular matrix preparation such as MATRIGEL(Collaborative Research), or removal of conditioned medium. Cells can beincubated in dishes and on cover slips coated with MATRIGEL to allowgellification and subsequently seeded onto the treated surface(Cardenas, A. M. et al., Neuroreport., 1999, 10:363–369).Differentiation can be induced by transfer to GM with 1% bovine serumand 10 μg/ml of both insulin and transferrin, wherein differentiatingmedia is F12/D supplemented with 1% bovine serum and 1% stock supplement(Liberona, J. L. et al., Muscle & Nerve, 1998, 21:902–909). Horse serumcan be utilized to increase fusion rate. Further differentiationprocedures and agents can be found, for example, in Caviedes, R. et al.,Brain Research, 1996, 365:259–268, where preconfluent cultures wereincubated in complete growth medium plus 2% dimethylsulfoxide for 10days, and in Arrigada, C. et al., Amino Acids, 2000, 18(4):363–373,where differentiation medium consisted of DMEM/Ham F12 nutrient mixture,supplemented with 2% adult bovine serum and 1% (v/v) of N3 supplementand 1% (v/v) Site+3 (SIGMA), and cells were allowed to differentiate for1 week.

Cells can be stimulated to differentiate by contact with one or moredifferentiation agents (e.g., trophic factors, hormonal supplements),such as forskolin, retinoic acid, putrescin-transferrin, cholera toxin,insulin-like growth factor (IGF), transforming growth factor (e.g.,TGF-α, TGF-β), tumor necrosis factor (TNF), fibroblast growth factor(FGF), epidermal growth factor (EGF), granulocyte macrophage-colonystimulating factor (GM-C SF), hepatocyte growth factor (HGF), hedgehog,vascular endothelial growth factor (VEGF), thyrotropin releasing hormone(TRH), platelet derived growth factor (PDGF), sodium butyrate, butyricacid, cyclic adenosine monophosphate (cAMP), cAMP derivatives (e.g.,dibutyryl cAMP, 8-bromo-cAMP) phosphodiesterase inhibitors, adenylatecyclase activators, prostaglandins, ciliary neurotrophic factor (CNTF),brain-derived neurotrophic factor (BDNF), neurotrophin 3, neurotrophin4, interleukins (e.g., IL-4), interferons (e.g., interferon-gamma),leukemia inhibitory factor (LIF), potassium, amphiregulin, dexamethasone(glucocorticoid hormone), isobutyl 3-methyulxanthine, somatostatin,lithium, and growth hormone.

The subject invention provides a ready source of cells for research,including pharmacological studies for the screening of various agents,and toxicologic studies for the cosmetic and pharmaceutical industries.The cells of the subject invention can be used in methods fordetermining the effect of a synthetic or biological agent on cells. Theterm “biological agent” refers to any agent of biological origin, suchas a virus, protein, peptide, amino acid, lipid, carbohydrate, nucleicacid, nucleotide, drug, pro-drug, or other substance that may have aneffect on cells, whether such effect is harmful, beneficial, orotherwise. Thus, the cells of the present invention can be used forscreening agonists and antagonists of compounds and factors that affectthe various metabolic pathways of a specific cell, for example. Thechoice of cell will depend upon the particular agent being tested andthe effects one wishes to achieve. For example, cells from a cardiacmuscle cell line can be incubated in a dose-escalation manner in vitroto evaluate changes in membrane potential, etc. Chemotherapies, such asthe administration of ADRIAMYCIN, are known to cause cardiac toxicity.Therefore, cardiac cell lines of the subject invention are useful fortesting such chemotherapies for cardiac toxicity. For example, the RCVCcell line of the subject invention described in Example 5 can be exposedto various synthetic or biological agents and the effects of the agentson the physiology of the cell can be determined by comparison ofphysiological criteria in a control (e.g. in the absence of the agents)(Caviedes, P. et al., J. Molec. & Cell Cardiol., 1993,25(1993):829–845). Further, sulfonamides induce toxicity of the pancreasacinar cells. Therefore, pancreatic acinar cell lines and other celllines of the subject invention would be useful for testing the toxicityof such agents. As shown in FIGS. 19A–B, 20A–C, 21A–B, and 22A–B, RCSN-3cells produced using the methods of the subject invention presentcharacteristic properties of neuronal dopaminergic cells in vitro,presenting apoptotic phenomena when exposed to pro-neurodegenerativeagents. Many drugs are known to induce liver damage. Therefore, toaddress this, a liver cell line of the subject invention can be used fortoxicity testing. A kidney cell line can be proliferated and usedsimilarly according to the methods of the subject invention.

The effects of synthetic or biological agents on the cells can beidentified on the basis of significant difference relative to controlcultures with respect to criteria such as the ratios of expressedphenotypes, cell viability and alterations in gene expression. Physicalcharacteristics of the cells can be analyzed by observing cellmorphology and growth with microscopy. Increased or decreased levels ofproteins, such as enzymes, receptors and other cell surface molecules,amino acids, peptides, and biogenic amines can be analyzed with anytechnique known in the art which can identify the alteration of thelevel of such molecules. These techniques include immunohistochemistry,using antibodies against such molecules, or biochemical analysis. Suchbiochemical analysis includes protein assays, enzymatic assays, receptorbinding assays, enzyme-linked immunosorbent assays (ELISA),electrophoretic analysis, analysis with high performance liquidchromatography (HPLC), Western blots, and radioimmune assays (RIA).Nucleic acid analysis, such as Northern blots and polymerase chainreaction (PCR) can be used to examine the levels of mRNA coding forthese molecules, or for enzymes which synthesize these molecules.

Alternatively, cells treated with these agents can be transplanted intoan animal, and their survival and biochemical and immunologicalcharacteristics examined as previously described.

Proliferated cells can be used as a platform for growing virus particlesfor vaccine production or other purposes. For example, human cervicalepithelium can be proliferated in culture and used to supportpapopavirus in the development of a vaccine. In addition, fetal kidneycells are commonly used for the production of several differentvaccines.

Cells proliferated by the methods of the subject invention can have anaturally occurring or induced defect, such that the cells provide an invitro model of disease. As described above with respect to normal cells,these cells can be used to test effects of synthetic or biologicalagents in a disease model. For example, the establishment of stable, invitro models of the nervous system will provide an important tool torapidly and accurately address various neurological disorders.Therefore, a cell line proliferated according to the methods of thesubject invention can be obtained having similar dysfunction mechanismsas the originating tissues, and which would serve as a model to studypotential therapies and/or further alterations of the cell function. Forexample, muscle isolated from Duchenne muscular dystrophy patients canbe used for investigating specific biochemical and genetic abnormalitiesassociated with that disease.

In addition, the cells of the subject invention can be used to generateantibodies for cell-specific proteins, and elucidate the interactionsbetween cell types and cell matrix components. Immune cells can beproliferated for administration to patients as immunotherapy. Forexample, B cell and T cell lines with specific anti-cancer propertiescan be proliferated and used for cell vaccine therapy (Couzin, J.Science, Sep. 20, 2002, 297:1973; Dudley M. E. et al. Science, Oct. 25,2002, 298:850–854). Furthermore, antibody producing cell lines directedagainst tumor necrosis factor can be utilized for treatment ofrheumatoid arthritis or psoriatic arthritis, and other autoimmunedisorders. Antibodies to cell-surface markers may be generated and usedto purify a subpopulation from a heterogenous population of cells usinga cell sorting system. Using membrane fragments of cells of the subjectinvention, monoclonal antibodies can be produced according to methodsknown in the art (Kohler et al., Nature, 1975, 256:495; Kohler et al.,Eur. J. Immunol., 1976, 6:511–519) and screened using a variety of celllines to identify antibodies that display cell specificity. In addition,cell-specific monoclonal antibodies can be used to purify cell-surfacemarkers and identify their function. Stem cells and precursor cells ofthe subject invention can be labeled, for example, usingβ-galactosidase, and their ontogeny followed in heterogenous cell andnutrient environments.

Once an immortalized cell line has been established, genetic materialfrom the cells can be used to construct cDNA libraries. Methods forpreparing cDNA libraries are well known in the art (Sambrook et al.,(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold SpringHarbor Laboratory Press, Cold Springs Harbor, N.Y.; Ausabel et al.,eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc.New York). By selecting cells at various stages of differentiation, thebiological functions that are associated with a specific stage in thedifferentiation pathway can be identified once a cDNA library isprepared from that cell's mRNA. The libraries can be used to clone novelfactors produced by specific cell types, such as differentiationfactors, growth hormones, and other cytokines and growth factors.

Cell lines prepared by the methods of the subject invention can also beused to prepare a protein library, which is complementary to the cDNAlibrary. Amino acid sequence information obtained from the libraryenables rapid isolation of cDNAs encoding proteins of interest. Couplingof protein and cDNA libraries also facilitates the targeted cloning ofsequences of particular interest. A protein library is prepared byextracting protein (total proteins or fractions of interest) from cellsaccording to known methods, and separating the proteins bytwo-dimensional gel electrophoresis, for example. Isolated proteins canthen be subjected to in situ digestion (e.g., tryptic digestion)followed by separation by micro-bore HPLC. The separated fragments canthen be analyzed by mass spectrometry. The resulting mass profile can besearched against a protein sequence database to infer protein identity.Unidentified peptides can be sequenced by Edman degradation. Theresulting cDNA and protein libraries are valuable sources of newproteins and the sequences encoding them.

Cell Products

Cells can be proliferated using the methods of the subject invention andthe cells' products harvested using methods known in the art. Variousbiomolecules produced by genetically modified or non-geneticallymodified cells that are proliferated using the methods of the subjectinvention can be harvested (e.g. isolated from the biomolecule-producingcells using methods known in the art) for various uses, such as theproduction of drugs and for pharmacological studies. Thus, using themethods of the subject invention, cells can be proliferated to producecontinuously growing cells and be used as biological “factories” toprovide the product of exogenous DNA and/or the natural product of thecells in vitro, or in vivo within an animal. The term “biomolecule”refers to any molecule or molecules that can be produced by cells. Suchbiomolecules include, but are not limited to, proteins, peptides, aminoacids, lipids, carbohydrates, nucleic acids, nucleotides, viruses, andother substances. Some specific examples of biomolecules include trophicfactors, hormones, and growth factors, such as brain-derived growthfactor (BDNF) and glial-derived neurotrophic factor (GDNF). For example,pituitary cells can be proliferated to produce growth hormone; kidneycells can be proliferated to produce plasminogen activator; bone cellscan be proliferated to produce bone morphogenetic protein (BMP) or otherproteins involved in bony fusions or prosthetic surgery (Urist, M. R.and Strates, B. S. J. Dent. Res. Suppl., 1971, 50:1392–1406; Boden, S.D. et al., Spine, 1995, 20:2633–2644; Boden, S. D. and Sumner, D. R.Spine, 1995, 20(Suppl. 24):1025–1125) and hepatitis-A antigen can beproduced from proliferated liver cells. Cells can be proliferated toproduce various viral vaccines and antibodies. Interferon, insulin,angiogenic factor, fibronectin and numerous other biomolecules can beproduced by proliferating cells to establish continuous cell lines. Thebiomolecules can be intracellular, transmembrane, or secreted by thecells, for example.

Administration of Cells

In another aspect, the subject invention concerns methods for treating avariety of disorders or traumatic injury by administering cells fromimmortalized cell lines of the invention to a patient (e.g., a human orother animal) in need thereof. Optionally, the proliferated cells can beisolated (removed from contact with) the proliferation factor of theinvention prior to their administration to a patient. Advantageously,because cells of the subject invention do not require the incorporationof an oncogene, and can be induced to arrest proliferation in vitro orin vivo, they can express a differentiated phenotype in vitro or invivo. The majority of cell lines of the subject invention arenon-tumorgenic in vivo. Therefore, non-tumorgenicity of a particularcell line can be determined using methods known in the art and the cellscan be administered to a patient in need thereof.

The cell lines of the subject invention can be administered as celltherapy to alleviate the symptoms of a wide variety of disease statesand pathological conditions, in various stages of pathologicaldevelopment. For example, cells of the subject invention can be used totreat acute disorders (e.g., stroke or myocardial infarction), andadministered acutely, subacutely, or in the chronic state. Similarly,the cells of the subject invention can be used to treat chronicdisorders (e.g., Parkinson's disease, diabetes, or muscular dystrophy),and administered preventatively and/or prophylactically, early in thedisease state, in moderate disease states, or in severe disease states.For example, the cells of the subject invention can be administered to atarget site or sites on or within a patient in order to replace orcompensate for the patient's own damaged, lost, or otherwisedysfunctional cells. This includes infusion of the cells into thepatient's bloodstream. The cells to be administered can be cells of thesame cell type as those damaged, lost, or otherwise dysfunctional, or adifferent cell type. For example, insulin-producing pancreatic isletbeta cells supplemented with other types of cells of the subjectinvention can be administered to the liver (Goss, J. A., et al.,Transplantation, Dec. 27, 2002, 74(12):1761–1766). As used herein,patients “in need” of the cells of the subject invention include thosedesiring elective surgery, such as elective cosmetic surgery.

The cells of the invention can be administered as autografts, syngeneicgrafts, allografts, and xenografts, for example. As used herein, theterm “graft” refers to one or more cells intended for implantationwithin a human or other animal. Hence, the graft can be a cellular ortissue graft, for example.

Proliferated cells can be administered to a patient by any method ofdelivery, such as intravascularly, intracranially, intracerebrally,intramuscularly, intradermally, intravenously, intraocularly, orally,nasally, topically, or by open surgical procedure, depending upon theanatomical site or sites to which the cells are to be delivered.Proliferated cells can be administered in an open manner, as in theheart during open heart surgery, or in the brain during stereotacticsurgery, or by intravascular interventional methods using cathetersgoing to the blood supply of the specific organs, or by interventionalmethods such as intrahepatic artery injection of pancreatic cells fordiabetics.

The cells of the subject invention can be administered to a patient inisolation or within a pharmaceutical composition comprising the cellsand a pharmaceutically acceptable carrier. As used herein, apharmaceutically acceptable carrier includes solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic agents, and thelike. Pharmaceutical compositions can be formulated according to knownmethods for preparing pharmaceutically useful compositions. Formulationsare described in a number of sources that are well known and readilyavailable to those of ordinary skill in the art. For example,Remington's Pharmaceutical Science (Martin E. W., Easton Pa., MackPublishing Company, 19^(th) ed.) describes formulations that can be usedin connection with the subject invention. Formulations suitable forparenteral administration, for example, include aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions that may include suspending agents and thickening agents. Itshould be understood that in addition to the ingredients particularlymentioned above, the formulations of the subject invention can includeother agents conventional in the art having regard to the type offormulation and route of administration in question.

The cells of the subject invention can be administered on or within avariety of carriers that can be formulated as a solid, liquid,semi-solid, etc. For example, genetically modified cells ornon-genetically modified cells can be suspended within an injectablehydrogel composition (U.S. Pat. No. 6,129,761) or encapsulated withinmicroparticles (e.g., microcapsules) that are administered to thepatient and, optionally, released at the target anatomical site (Read T.A. et al., Nature Biotechnology, 2001, 19:29–34, 2001; Joki T. et al.,Nature Biotechnology, 2001, 19:35–38; Bergers G. and Hanahan D., NatureBiotechnology, 2001, 19:20–21; Dove A. Nature Biotechnology, 2002,20:339–343; Sarkis R. Cell Transplantation, 2001, 10:601–607).

Microcapsules can be composed of various polymers and, in addition tocells, their contents can include enzymes and other materials.Preferably, the microcapsules are prepared in such a way as to preventtheir contents from leaking out and potentially causing an immunologicalreaction, while permitting nutrients and metabolites to exchange freely.Microencapsulation of hepatocytes has been used to prepare so-called“bio-artificial liver assist devices” (BLAD). The high surface-to-volumeratio of a spherical microcapsule facilitates maximal transport ofnutrients, gases, or metabolites exchange across the membrane. Inaddition, encapsulation of living cells allows better control of themicroenvironment for optimal cellular functions via selection ofsuitable substrates and incorporation of controlled release features, asdescribed below. Such devices can be used to deliver various types ofcells proliferated according to the methods of the subject invention.Microcapsules can carry a payload of more than one type of cell. Forexample, islet cells can be encapsulated with Sertoli cells andadministered to a patient.

Carriers are preferably biocompatible and optionally biodegradable.Suitable carriers include controlled release systems wherein the cellsand/or the biological factors produced by the cells are released fromthe carrier at the target anatomic site or sites in a controlled releasefashion. The mechanism of release can include degradation of the carrierdue to pH conditions, temperature, or endogenous or exogenous enzymes,for example.

The cells of the invention can be administered in or on variousscaffolds, such as synthetic or biological tissue scaffolds (Griffith G.and Naughton G., Science, 2002, 295:1009–1013; Langer R., Stem CellResearch News, Apr. 1, 2002, pp. 2–3). Porous scaffold constructs can becomposed of a variety of natural and synthetic matrices, such asbiominerals (e.g., calcium phosphate) and polymers (e.g., alginate) thatare optionally cross-linked, and serve as a template for cellproliferation and ultimately tissue formation. Three-dimensional controlof pore size and morphology, mechanical properties, degradation andresorption kinetics, and surface topography of the scaffold can beoptimized for controlling cellular colonization rates and organizationwithin an engineered scaffold/tissue construct. In this way, themorphology and properties of the scaffold can be engineered to providecontrol of the distribution of bioactive agents (e.g., proteins,peptides, etc.) and cells. In addition to use as vehicles for deliveryof the proliferated cells, scaffolds can be utilized to grow the cellsin vitro. Optionally, cells can be proliferated on the scaffoldsthemselves using the methods of the subject invention.

Scaffolds can contain interconnecting networks of pores and facilitateattachment, proliferation, and biosynthesis of cartilaginous matrixcomponents, where desired. For example, synthetic or biologicalscaffolds carrying bone cells, such as chondrocytes, of the subjectinvention can be administered to a patient in need thereof. Chitosanscaffolds, which are biocompatible and enzymatically degraded in vivo,can be seeded with chondrocytes proliferated according to the methods ofthe subject invention and implanted. An alginate scaffold can befabricated in the shape of a heart valve, seeded with proliferated cellsof the invention, and implanted within a patient in need thereof.Because alginate does not naturally provide anchorage points for cells,in order to facilitate cell attachment, the peptide sequence R-G-D(Arginine-Glycine-Aspartic acid) can be utilized to act as a ligand forcell integrins and can be linked to alginate.

The cells of the subject invention are preferably administered to apatient in an amount effective to provide a therapeutic benefit. A“therapeutically effective amount” is that amount effective to treat apathological condition. For purposes of the subject invention, the terms“treat” or “treatment” include preventing, inhibiting, reducing theoccurrence of and/or ameliorating the physiological effects of thepathological condition to be treated. Preferably, the cells areadministered to the patient in an amount within the range of about 10⁴to about 10¹⁰ cells. More preferably, the cells are administered to thepatient in an amount within the range of about 10⁷ to about 10¹⁰ cells.Doses of cells can be determined by one of ordinary skill in the art,with consideration given to such factors as cell survival rate followingadministration, the number of cells necessary to induce a physiologicresponse in the normal state, and the species of the patient.

Mammalian species which benefit from the disclosed methods of treatmentinclude, and are not limited to, apes, chimpanzees, orangutans, humans,monkeys; domesticated animals (e.g., pets) such as dogs, cats, guineapigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. As usedherein, the term “patient” refers to a recipient of the cells of thesubject invention. For example, suitable patients include the foregoingmammalian species.

The cell lines of the subject invention have advantages over other cellsknown in the art that are currently being utilized for transplantationpurposes. The cell lines of the subject invention can be of humanorigin. The subject invention makes it possible to proliferate otherwisenondividing or very slowly dividing cells (e.g., dopaminergic neurons orinsulin-producing beta-cells), which is an important aspect for theproduction of biological molecules and from a cell therapy perspective.Therefore, such cells can serve as clinical allografts from animmunological perspective, are available in adequate quantities, can bemanufactured using good manufacturing processes, and can be producedfree of contaminants from other cell types that might contaminatenonproliferated cells or cells derived from stem cells. The cells can becryopreserved, are available for elective surgery, can be standardizedand characterized before use. The cells can be rendered non-dividing andcan have known HLA types that may facilitate advantageous immunologicmatching or intentional mismatching, or allow for the production ofmultiple immunologically matched cell lines.

Using the methods of the subject invention, multiple identical celllines from different donors can be created that vary only in theirimmunologic expression of surface antigens, based on the donor fromwhich they are derived. Therefore, a cell line that has a closeimmunologic match to a particular potential recipient can be moreclosely customized. It is therefore possible using this technique tomake a donor-specific cell line that is immunologically matched to thedonor, or to make intentionally mismatched but related cell lines if sodesired, for example, for the transplantation in genetic disorders whereit may be desired to have a genetically mismatched cell line. Based uponthe human population's various histotypes as well as currentimmunosuppression techniques, it has been determined that no more thantwelve cell lines would be a sufficient donor source for the majority ofABO and type II antigen combinations found in 70%–80% of all Anglo Saxonpatients in American and Europe. Furthermore, if the donor is type O,less than eleven cell lines would be a sufficient donor source.

For systemic transplants (e.g., pancreas), multiple different cell linescan be produced. Thus, a cell line that is similar to each patient canbe available if enough cell lines are available, minimizing rejectionrisk. Additionally, if a patient requires a second transplant, a cellline that is different immunologically from the first can betransplanted, and there will not be induction of second-set rejection ofthe first cell line transplant. Furthermore, if the cell lines are notexactly immunologically the same as the patient, then discontinuation ofthe immunosuppression will cause graft rejection. This strategy can beused for elimination of toxicity if too many cells are transplanted orunexpected adverse events develop. In the CNS, immunologically unmatchedcells will not be rejected, even in the absence of immunosuppression.Thus, for safety purposes, it may be necessary to be able to reject aneural graft if toxicity develops. For this, it would be necessary togrow a neural and skin cell line from the same donor (or, for example,dopamine neuron, retinal pigment epithelial cell, kidney cell, and skincell, if these are to be transplanted into the CNS). Transplantation ofthe immunologically identical skin cell line orthotopically (into theperiphery) will induce second set rejection of the neural graft (Freed,W. J., Biological Psychiatry [1983] 18:1204–1267; Nicholas, M. K. et al.J. Immunology [1987] 139:2275–2283; Mason D. W. et al. Neuroscience[1986] 19:685–694).

Combinations of cell lines can be co-administered to enhance therapeuticpotential. For example, a trophic factor-producing cell line can beco-administered with a neuronal cell line. An insulin-secreting cellline and a glucagon cell line, with or without a pancreatic ductal cellline, can be co-administered for the treatment of diabetes. Methods ofco-administration may include production of cell lines together (e.g.,in a roller flask), or individually in separate batches that are mixedbefore implantation. Ratios and volumes of cells proliferated in culturecan have some influence on efficacy and viability of cells in vitro orin vivo. A Sertoli cell line can be co-administered with a cell line ofanother species (as a xenograft), such that the Sertoli cells providelocal immunosuppression of the xenograft. Sertoli cells can providelocal immunosuppression for allografts (in addition to xenografts)transplanted systemically, such that immunosuppression may not benecessary (or only reduced amounts may be necessary).

Using normally non-dividing cell lines from the pancreas, the subjectinvention can provide a treatment for patients suffering from type 1 ortype 2 diabetes, pancreatitis, post resection, or any conditionrequiring replacement of the pancreas. For example, human alpha and/orbeta cells in the pancreas can be proliferated using the methods of thesubject invention, for the purpose of replacing both glucagon and/orinsulin-secretion properties of the pancreas. Entire pancreas isletscontaining a repertoire of pancreas cell types (e.g., α cells, β cells,δ cells, pancreatic polypeptide-producing cells) can also beproliferated and administered.

Further, proliferation of cells from other organs and tissues can beperformed, including, but not limited to, cells of blood vessels, skin,fat, chondrocyte/bone, tendon, ligaments, and cartilage. Skin cells canbe useful, for example, in treating chronic ulcers (e.g., decubitus ordiabetic foot ulcers); tendon, ligament, and cartilage cells can beuseful for treatment of degenerative diseases, osteoarthritis, andrheumatoid arthritis, as well as for orthopedic reconstructions. Inaddition, cardiac muscle or heart valve cells can be proliferated usingthe methods of the subject invention and administered to a patientfollowing myocardial infarction or other causes of damage to heartmuscle or valve. Liver cells can be proliferated for treatment ofhepatitis or liver failure. Corneal cells can be proliferated forcorneal transplants. Neuroendocrine chromaffin cells of the adrenalmedulla can be proliferated using methods of the subject invention.Neuroendocrine chromaffin cells secrete opioid peptides, catecholamine,and several neuropeptides, including somatostatin, neuropeptide Y, andneurostatin, and can be administered to a patient (e.g., into thesubarachnoid space, spinal cord, or brain) for acute or chronic painconditions, such as inflammatory arthropathies and neuropathic pain.Sympathetic chain adrenergic neuron cells can also be proliferated.Chondrocytes can be proliferated for patients with arthrosis. Forexample, such cells can be obtained from the patient's other joints,proliferated to produce a chondrocyte cell line using the methods of thesubject invention, and subsequently administered to the patient'sdiseased or damaged joints.

Hepatocytes of the subject invention can be administered directly to thepatient's liver. However, in an alternative embodiment, hepatocytesproliferated using the methods of the subject invention can be placedwithin a device to be administered into the patient's circulatory systemso that the cells can perform liver function at sites anatomicallyseparate from the patient's liver (Sarkis R. et al., CellTransplantation, 2002, 10:601–607). In addition to the administration ofliver cells as therapy for hepatitis and metabolic disorders, thesecells can also be administered for treatment of acute or chronic liverfailure, either as a bridge for a patient awaiting liver transplantationor as a definitive therapy requiring no further liver transplantation(Kobayashi, N. and Tanaka, N., Cell Transplantation, 2002, 11:417–420).Further, liver cells can be administered as a cancer treatment forpatients who require curative doses of hepatic radiation.

According to methods of the subject invention, it is also possible toproliferate hematogenous and lymphoid cells for the treatment of cancerssuch as lymphoma, myeloma, and leukemia, as well as for bone marrowtransplantation purposes. Further, the proliferation of human dendritic(blood-derived) cells can be used in the restoration, repair, oraugmentation of the immune system in immunotherapy, either in a diseasestate, such as in HIV, an autoimmune disorder, or cancer, or followingchemotherapy or radiation therapy. Adrenal cortical tissue can also beproliferated for addressing adrenocortical insufficiency, such as inAddison's disease. Proliferated pituitary tissue is useful for pituitaryinsufficiency, such as for specific hormonal needs (i.e., TSH,prolactin, ACTH, or other hormone-producing cells from the pituitary),which is useful in transplantation following menopause, hysterectomy, orchemotherapy. Proliferated ovarian cells are also useful in similarsituations. Further, egg cells can be proliferated for a variety ofuses, such as cloning, research, or in vitro fertilization. Pulmonarymesenchymal cell can be proliferated and administered for treatment ofdiseases of the lung including cystic fibrosis and emphysema. Cells ofthe vocal cords or stem cells can be proliferated and administered forthe repair vocal cords or production of vocal cord organs. Likewise,thymus cells or stem cells can be proliferated for the production ofimmune cells, such as T cells, or the repair or production of thymusorgans.

It has been observed that bone marrow transplants will induce tolerancebefore kidney transplantation of allogeneically related kidney donor.Therefore, using the methods of the subject invention, a bone marrowhematogenous cell line derived from the same donor as an organ cell line(e.g., pancreatic, heart, etc.) can be utilized to induce tolerance ofthe proliferated bone marrow hematogenous cell line (Dove A., NatureBiotechnology, 2002, 20:339–343).

Retinal cells can also be proliferated for transplantation to treatpathological conditions of the eye such as retinitis pigmentosa (arhodopsin defect), ischemic retinopathy, and macular degeneration. Humanretinal pigment epithelial cells and human iris pigment epithelial cellscan be proliferated and administered to a patient for the restoration ofvision or Parkinson's disease. Embryonic or other stem cells can beproliferated and administered subretinally to rescue photoreceptor cellsfrom degeneration, for example.

According to the methods of the subject invention, neutrophils can beproliferated and be intravenously administered for treatment of septicshock in children, for example. This treatment can be utilized in casesof sepsis, as well as cancer patients that are immunocompromisedfollowing chemotherapy. Conventionally, it is not possible to obtain asufficient amount of neutrophils for use in adult patients. For example,current protocols exist where neutrophil production is stimulated inpatients with bone marrow suppression using colony stimulating factors,which is very expensive. Advantageously, using the methods of thesubject invention, a neutrophil cell line can be proliferated andutilized for treatment of adult (and pediatric) sepsis patients. Itshould be understood that, even if the cells are rejected, it isexpected that they would attack the infectious agents responsible forinduction of septic shock before the cells are rejected.

The methods of the subject invention contemplate intracerebral graftingof donor cells to a region of the CNS, such as a region having sustaineddefect, disease, or trauma. Neural transplantation or “grafting”involves transplantation of cells into the central nervous system orinto the ventricular cavities, or subdurally onto the surface of thehost brain. Conditions relevant to successful transplantation include:(i) viability of the implant; (ii) retention of the graft at theappropriate site of transplantation; (iii) minimum amount ofpathological reaction at the site of transplantation; (iv) maintenanceof specific cell function; (v) prevention of immune reaction; and (vi)provision of trophic support and vascular supply. Parameters relevant tothe above conditions include source of tissue, donor age, number ofdonors, distribution of grafted tissue, site of implantation, method ofcell storage, and type of graft (cell suspension or solid).

Methods for transplanting various nerve tissues as allografts andxenografts have been described previously (Freeman T. B. et al.,Progress in Brain Research, 1988, Chapter 61, 78:473–477; Freeman T. B.et al., Parkinson's Disease: Advances in Neurology, 2001, Chapter 46,86:435–445; Freeman T. B. et al., Annals of Neurology, 1995,38(3):379–387; Freeman T. B. et al., Progress in Brain Research, 2000,Chapter 18, 127:405–411; Olanow C. W. et al. The Basal Ganglia and NewSurgical Approaches for Parkinson's Disease, Advances in Neurology,1997, 74:249–269; Bjorklund et al., Neural Grafting in the MammalianCNS, 1985, p. 709, Elsevier, Amsterdam; Das G. D., Neural Grafting inthe Mammalian CNS, 1985, Chapter 3, p. 23–30, Elsevier, Amsterdam).These procedures include intraparenchymal transplantation, i.e., withinthe host brain tissue (as compared to outside the brain orextraparenchymal transplantation) achieved by injection or deposition oftissue within the host brain so as to be opposed to the brain parenchymaat the time of transplantation.

Methods for intraparenchymal transplantation include, for example: (i)injecting the donor cells within the host brain parenchyma (e.g.,stereotactically, using image guidance, and/or with a catheter attachedto a pump, such as a MEDTRONIC system); and (ii) preparing a cavity bysurgical means to expose the host brain parenchyma and then depositingthe graft into the cavity. Such methods provide parenchymal appositionbetween the graft and host brain tissue at the time of grafting, andboth facilitate anatomical integration between the graft and host braintissue.

Alternatively, the graft can be place into the cerebral spinal fluid(CSF), either by open surgical injection, intraventricularly via aneedle or ventricular reservoir, into the lumbar subarachnoid spaceusing a lumbar puncture, or into any CSF site using a pump and acatheter (e.g., MEDTRONIC). These methods would lend themselves torepeated administration over time, to the CSF or to the brain. Graftingto the ventricle may be accomplished by injection of the donor cells orby growing the cells in a substrate such as 3% collagen to form a plugof solid tissue which may then be implanted into the ventricle toprevent dislocation of the graft. For subdural grafting, the cells maybe injected around the surface of the brain after making a slit in thedura. Injections into selected regions of the host brain can be made bydrilling a hole and piercing the dura to permit the needle of amicrosyringe to be inserted. The microsyringe can be mounted in astereotactic frame and three-dimensional stereotactic coordinates areselected for placing the needle into the desired location of the brainor spinal cord. Image guidance methods can also be utilized. The cellsof the subject invention can also be introduced into the putamen,caudate nucleus, pallidum, nucleus basalis, hippocampus, cortex,cerebellum, subcortical white matter, other regions of the brain, aswell as the spinal cord using intravascular technique (Amar A. P. et al.Neurosurgery [2003] 52:402–413).

Many of the aforementioned cell lines produce trophic factors, includingthe Sertoli cell line, glial cell lines, and many of the aforementionedneuronal cell lines. Retinal pigment epithelial (RPE) cells, irispigment epithelial (IPE) cells, kidney cells, and hNT cells, amongothers, produce neurotrophic factors. These cell lines are useful fortheir trophic factor production properties for the treatment ofneurologic disorders, including, but not limited to, Parkinson'sdisease, Huntington's disease, amyotrophic lateral sclerosis (ALS), andstroke. For example, cells from immortalized cell lines of the subjectinvention can be administered to a patient to supplement the pool ofdopaminergic neurons and to reinstate the dopaminergic input in thestriatum. In addition, cells that natively or are modified to secreteciliary neurotrophic factor (CNTF) and/or brain-derived neurotrophicfactor (BDNF) can be proliferated and administered to a patient fortreatment of Huntington's disease. Numerous trophic factors remain to beidentified that play an important role in the development or maintenanceof various cells in the body, both in normal and pathophysiologicalstates. Using the methods of the subject invention, proliferation ofcells that produce these factors is contemplated for both therapeuticand manufacturing purposes, as well as for investigational andlaboratory purposes.

Adult stem cells or nondividing cells from a recipient with a disease ofa particular organ can be proliferated using the methods of the subjectinvention for transplantation purposes. These cells can be isogenic(immunologically matched, donor-specific) with the particular patient.For example, if a pancreas has to be removed following an episode ofpancreatitis, a similar piece of tissue can be proliferated for thatindividual patient and implanted without the need for immunosuppression.Heart muscle cells can likewise be proliferated and administered toreplace damaged heart muscle in a patient suffering from congestiveheart failure. This is particularly advantageous in non-life-threateningdisorders, wherein the risk of immunosuppression is a concern. Suchdiseases include proliferation of corneal tissue for cornea replacement;tendon, ligament, and cartilage proliferation for orthopedic proceduresor for treatment of degenerative disorders; ovarian cortical cellproliferation for hormonal replacement following menopause orhysterectomy; and keratinocyte and collagen preparations for diabeticulcers, for example.

Genetically Modified Cells

The methods of the subject invention also contemplate the administrationof genetically modified cells alone or in combinations with differenttypes of cells. Thus, genetically modified cells of the invention can beco-administered with other cells, which can include genetically modifiedcells or non-genetically modified cells. Genetically modified cells mayserve to support the survival and function of the co-administered cells,for example.

The term “genetic modification” as used herein refers to the stable ortransient alteration of the genotype of a cell of the subject inventionby intentional introduction of exogenous nucleic acids by any meansknown in the art (including for example, direct transmission of apolynucleotide sequence from a cell or virus particle, transmission ofinfective virus particles, and transmission by any knownpolynucleotide-bearing substance) resulting in a permanent or temporaryalteration of genotype. The nucleic acids may be synthetic, or naturallyderived, and may contain genes, portions of genes, or other usefulpolynucleotides. The term “genetic modification” is not intended toinclude naturally occurring alterations such as that which occursthrough natural viral activity, natural genetic recombination, or thelike. However, such naturally altered cells can also be proliferatedaccording to the methods of the subject invention.

Exogenous nucleic acids can be introduced into a cell of the subjectinvention by viral vectors (retrovirus, modified herpes virus, herpesvirus, adenovirus, adeno-associated virus, and the like) or direct DNAtransfection (lipofection, calcium phosphate transfection, DEAE-dextran,electroporation, and the like), for example.

In another embodiment, the cells are derived from transgenic animals,and thus are in a sense already genetically modified. There are severalmethods presently used for generating transgenic animals. A typicaltechnique is direct microinjection of DNA into single-celled fertilizedeggs. Other methods include retro-viral-mediated transfer, or genetransfer in embryonic stem cells. These techniques and others aredetailed by Hogan et al. in Manipulating the Mouse Embryo, A LaboratoryManual (Cold Spring Harbor Laboratory Ed., 1986).

The genetically modified cells (so called “designer cell lines”) of thesubject invention can be administered to a patient for cell/genetherapy, e.g., for in vivo delivery of various biomolecules, such as thetrophic factors described above. Alternatively, the genetically modifiedcells can be used as biological “factories” to provide the product ofthe exogenous DNA and/or the natural product of the modified cells invitro, or in vivo within an animal. Genetically modified cells can bestem cells or non-stem cells, for example.

The cells of the subject invention, whether genetically modified ornon-genetically modified, can be co-administered with therapeutic agentsuseful in treating defects, trauma, or diseases, such as growth factors,antibiotics, or neurotransmitters.

The cells of the subject invention can be genetically modified (e.g.,genetically engineered) to produce a vast array of biologically activemolecules, such as cytokines, growth factors, antigens, receptors,glycoproteins, and enzymes, before, during, or after proliferation.Cells can be genetically modified to produce toxins, drugs forcell-based delivery, chemotherapy, neurotransmitters, and otherbiomolecules. Cells can be genetically modified to include regulators,inducible promoters, tissue-specific promoters, on-off genes, or suicidegenes. Exogenous genes that interfere with oxidative stress (e.g.,glutamate transporter) could be added to cells. B cells and T cells canbe genetically modified to make monoclonal antibodies with targets forspecific cancer cells, or against tumor necrosis factor (TNF), fortreatment of rheumatoid arthritis or psoriatic arthritis.

Genetically modified cell lines can include more than one geneticconstruct. For example, a dopamine cell line can be constructed fromembryonic stem cells, hNT neurons, or some other source. A secondaryconstruct for glial-derived neurotrophic factor (GDNF), a potent factorfor dopamine cell lines, can be added to the genetically modifieddopamine cell line. The modified cell line can then be proliferatedusing the methods of the subject invention. Similarly, a secondaryconstruct encoding antiapoptotic agents can be added. For example,genetic constructs encoding caspase inhibitors or interleukins canbenefit a cell's function and survival.

Furthermore, sonic hedgehog (Shh) and FGF-8 are required for theinduction of midbrain dopaminergic neurons during normal development,and the combination of Shh and FGF-8 can induce neurons with adopaminergic phenotype in ectopic regions along the anterior neural tube(Ye, W. et al., Cell, 1998, 93:755–766). Cells of the subject invention(e.g., fibroblasts) can be genetically modified to produce Shh and/orFGF-8 for therapeutic, manufacturing, or research purposes. For example,such genetically modified cells can be administered to a patient tosignificantly increase the number of surviving co-administereddopaminergic neurons (Yurek, D. M. et al., Cell Transplantation, 2001,10:665–671).

Stem cells can be genetically modified, then subsequently proliferatedusing the methods of the subject invention. Alternatively, stem cellscan be proliferated using the methods of the subject invention, thensubsequently genetically modified.

Non-stem cells (e.g., specialized or mature cells, and their precursoror progenitor cells) can be genetically modified, then subsequentlyproliferated using methods of the subject invention. Alternatively,non-stem cells can be proliferated using the methods of the subjectinvention, then subsequently genetically modified.

Proliferation Factor and Receptor

There are at least two possible mechanisms for the activity of the tumorcell line proliferation factor described herein, which are notnecessarily mutually exclusive. These mechanisms include, for example,phosphorylation of cyclin-dependent kinases (CDKs) or inhibition CKIs(CDK inhibitors); and/or interaction with telomerase or other DNA repairmechanisms (e.g., ligases), impairing normal DNA repair withoutcompromising function. A further aspect of the subject inventionincludes methods of modulating the growth cycle of a cell or cells.Possible target cells include those described herein with respect toother methods of the subject invention. Modulation of a cell's growthcycle can be carried out by contacting or otherwise exposing the cellsto the tumor cell line proliferation factor (including biologicallyactive fragments or analogues thereof), agonists of the proliferationfactor receptor, or functional antagonists of the proliferation factorreceptor, such as antagonistic antibodies. Such agonists and antagonistsmay operate directly or indirectly on the proliferation factor receptor,and/or within the proliferation pathway.

The UCHT1 proliferation factor and its receptor are merely exemplary ofother tumor cell line proliferation factors and correspondingproliferation factor receptors of the subject invention. Thus, thesubject invention also includes variant or equivalent tumor cell lineproliferation factors and proliferation factor receptors, such as thehomologous human proliferation factor and proliferation factor receptor.Variant or equivalent tumor cell line proliferation factors andreceptors (and nucleotide sequences coding for equivalent proliferationfactors and receptors) have the same or similar activities to the UCHT1proliferation factor and receptor. Equivalent proliferation factors andproliferation factor receptors will typically have amino acid homologywith the exemplified UCHT1 proliferation factor and receptor,respectively. This amino acid identity will typically be greater than60%, preferably greater than 75%, more preferably greater than 80%, morepreferably greater than 90%, and can be greater than 95%. Theseidentities are determined using standard alignment techniques. The aminoacid homology will be the highest in critical regions of theproliferation factor and receptor which account for biological activityor are involved in the three-dimensional configuration which ultimatelyis responsible for the biological activity. In this regard, certainamino acid substitutions and/or deletions are acceptable and can beexpected if these substitutions and deletions are in regions which arenot critical to activity or are conservative amino acid substitutions ordeletions which do not affect the three-dimensional configuration of themolecule. For example, amino acids can be placed in the followingclasses: non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby an amino acid of one class is replaced withanother amino acid of the same type fall within the scope of the subjectinvention so long as the substitution does not completely eliminate thebiological activity of the proliferation factor or proliferation factorreceptor; however preferred substitutions are those which result in theretention of most or all of the biological activity of the proliferationfactor or proliferation factor receptor. Table 1 provides a listing ofexamples of amino acids belonging to each class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Non polar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not completelyeliminate the biological activity of the receptor; however, preferredsubstitutions are those which result in the retention of most or all ofthe biological activity of the proliferation factor or proliferationfactor receptor. The use of polynucleotide probes is well known to thoseskilled in the art. In one specific example, a cDNA library for tumorcells (e.g., a thyroid tumor cell line) can be created by routine means,and DNA of interest can be isolated from the cDNA library.Polynucleotides of the subject invention can be used to hybridize withDNA fragments of the constructed cDNA library, allowing identificationof and selection (or “probing out”) of the genes of interest, i.e.,those nucleotide sequences which hybridize with the probes of thesubject invention and encode polypeptides having proliferation factoractivity or proliferation factor receptor activity. The isolation ofthese genes can be performed by a person skilled in the art having thebenefit of the instant disclosure, using techniques which are well-knownin the molecular biology art.

Thus, it is possible, without the aid of biological analysis, toidentify polynucleotide sequences encoding tumor cell line proliferationfactors and corresponding receptors. Such a probe analysis provides arapid method for identifying genes encoding proliferation factors andproliferation factor receptors from a wide variety of hosts. Theisolated genes can be inserted into appropriate vehicles which can thenbe used to transform a suitable host. The presence of genes encoding theproliferation factors and proliferation factor receptors of the subjectinvention can be determined in a variety of hosts, including cells otherthan those of tumor cell lines.

Various degrees of stringency of hybridization can be employed. The moresevere the conditions, the greater the complementarity that is requiredfor duplex formation. Severity of conditions can be controlled bytemperature, probe concentration, probe length, ionic strength, time,and the like. Preferably, hybridization is conducted under moderate tohigh stringency conditions by techniques well known in the art, asdescribed, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes,Stockton Press, New York, N.Y., pp. 169–170.

Examples of various stringency conditions are provided herein.Hybridization of immobilized DNA on Southern blots with ³²P-labeledgene-specific probes can be performed by standard methods (Maniatis etal. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York). In general, hybridization and subsequent washescan be carried out under moderate to high stringency conditions thatallow for detection of target sequences with homology to the exemplifiedpolynucleotide sequence. For double-stranded DNA gene probes,hybridization can be carried out overnight at 20–25° C. below themelting temperature (Tm) of the DNA hybrid in 6×SSPE, 5× Denhardt'ssolution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature isdescribed by the following formula:Tm=81.5° C.+16.6Log [Na+]+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs(Beltz et al. (1983) Methods of Enzymology, R. Wu, L. Grossman and K.Moldave [eds.] Academic Press, New York 100:266–285).

-   -   Washes are typically carried out as follows:    -   (1) twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS        (low stringency wash);    -   (2) once at Tm −20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS        (moderate stringency wash).

For oligonucleotide probes, hybridization can be carried out overnightat 10–20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE,5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes can be determined by the following formula:Tm(° C.)=2(number T/A base pairs)+4(number G/C base pairs)(Suggs, S. V., et al. (1981) ICN-UCLA Symp. Dev. Biol. Using PurifiedGenes, D. D. Brown [ed.], Academic Press, New York, 23:683–693).

-   -   Washes can be carried out as follows:    -   (1) twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS        (low stringency wash);    -   (2) once at the hybridization temperature for 15 minutes in        1×SSPE, 0.1% SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment >70 or so bases in length, the followingconditions can be used:

Low: 1 or 2X SSPE, room temperature Low: 1 or 2X SSPE, 42° C. Moderate:0.2X or 1X SSPE, 65° C. High: 0.1X SSPE, 65° C.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

As used herein, the terms “proliferate” and “propagate” are usedinterchangeably to refer to cell division. In the context ofproliferating cells by contacting or otherwise exposing the cells to atumor cell line proliferation factor, a tumor cell line that producessuch a proliferation factor, or a composition containing a tumor cellline proliferation factor (e.g., conditioned medium), it is intendedthat proliferation can include proliferation to the point of productionof a continuous cell line (e.g., immortalization, non-neoplastic, ornon-malignant transformation). The proliferation factors of the subjectinvention have a pro-proliferative effect on cells contacted with thefactor. When contacted with the factor, the cells are induced to attaina proliferation rate that is higher than the cells' normal proliferationrate in vitro, thus increasing the cells' potential for proliferation.

As used herein, the term “culture” is used to denote the maintenance orcultivation of cells in vitro including the culture of single cells.Cultures can be cell, tissue, or organ cultures, depending upon theextent of organization.

As used herein, the term “cell line” is used to refer to cells whichhave arisen from a primary culture and capable of successful subculture.

As used herein, the term “continuous cell culture” or “continuous cellline” is used to refer to a culture or cell line which is capable of anunlimited number of population doublings; often referred to as animmortal cell culture or cell line. Such cells may or may not expressthe characteristics of in vitro neoplastic or malignant transformation.This is antithesis of a finite cell culture or cell line, which iscapable of only a limited number of population doublings after which theculture or cell line ceases proliferation (i.e., in vitro senescence).

As used herein, the term “immortalization” refers to the attainment by afinite cell culture, whether by perturbation or intrinsically, of theattributes of a continuous cell line. An immortalized cell line is notnecessarily one which is neoplastically or malignantly transformed.

As used herein, the term “isolated” means removal from its nativeenvironment, and can include removal from its immediate nativeenvironment. As used herein, the term “isolated factor” or “isolatedproliferation factor” indicates that the factor has been isolated fromthe tumor cell line (e.g., the UCHT1 cell line) that produces it.

As used herein, the term “differentiated” refers to those cells thatmaintain in culture all, or a substantial amount of, their specializedstructure and function typical of the cell type in vivo. Partiallydifferentiated cells maintain less than a substantial amount of theirfull complement of specialized structure and/or function.

As used herein, the term “stem cell” is an unspecialized cell that iscapable of replicating or self renewal, and developing into specializedcells of a variety of cell types. The product of a stem cell undergoingdivision is at least one additional stem cell that has the samecapabilities of the originating cell. For example, under appropriateconditions, a hematopoietic stem cell can produce a second generationstem cell and a neuron. Stem cells include embryonic stem cells (e.g.,those stem cells originating from the inner cells mass of theblastocyst) and adult stem cells (which can be found throughout the moremature animal, including humans). As used herein, stem cells areintended to include those stem cells found in animals that have maturedbeyond the embryonic stage (e.g., fetus, infant, adolescent, juvenile,adult, etc.). The list of tissues reported to contain stem cells isgrowing and includes, for example, bone marrow, peripheral blood, brain,spinal cord, umbilical cord blood, amniotic fluid, placenta, dentalpulp, blood vessels, skeletal muscle, epithelia of the skin anddigestive system, cornea, retina, liver, and pancreas.

As used herein, the term “progenitor cell” (also known as a precursorcell) is unspecialized or has partial characteristics of a specializedcell that is capable of undergoing cell division and yielding twospecialized cells. For example, a myeloid progenitor/precursor cell canundergo cell division to yield two specialized cells (a neutrophil and ared blood cell).

As used herein, the term “phenotype” refers to all the observablecharacteristics of a cell (or organism); its shape (morphology);interactions with other cells and the non-cellular environment (e.g.,extracellular matrix); proteins that appear on the cell surface (surfacemarkers); and the cell's behavior (e.g., secretion, contraction,synaptic transmission).

As used herein, the terms “administer”, “apply”, “treat”, “transplant”,“implant”, “deliver”, and grammatical variations thereof, are usedinterchangeably to provide cells of the subject invention to a patient.

As used herein, the term “co-administration” and variations thereofrefers to the administration of two or more agents simultaneously (inone or more preparations), or consecutively.

All patents, patent applications, and publications referred to or citedherein are incorporated by reference in their entirety, including anyfigures, tables, nucleic acid sequences, amino acid sequences, ordrawings, to the extent they are not inconsistent with the explicitteachings of this specification.

Materials and Methods

Establishment of RCSN-3 cell line. The RCSN-3 cell line was derived fromthe substantia nigra of 4 month old normal Fisher 344 rats. The cellsused to establish primary cultures were immortalized by exposing them tomedia conditioned by UCHT1 cells (as shown in FIG. 1). For standardculture conditions, the cells were kept in feeding medium consisting ofDMEM/Ham F12 nutrient mixture (1:1) (SIGMA Chemical Co., Saint Louis,Mo., USA) modified to contain 6 g/l glucose, 10% bovine serum, 2.5%fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin (SIGMA)supplemented with 10% (v/v) with UCHT1 conditioned medium. The cultureswere maintained in an incubator at 37° C. with 100% humidity and anatmosphere of 10% CO₂ and were monitored routinely for the appearance oftransformation foci or morphological changes. After 10 weeks in culture,transformation foci were evident. The cultures were expanded and partwas cryopreserved in liquid nitrogen. The cell line was cloned bydilutional culturing, giving rise to the clonal line RCSN-3. Cells werepassaged at confluence with trypsinization (1% trypsin, GIBCO, GrandIsland, N.Y., USA). For standard growth conditions, RCSN-3 cells werecultured in feeding medium. Media was renewed completely twice a week.For differentiation, the cells were kept in a media consisting ofDMEM/Ham F12 nutrient mixture, supplemented with 2% adult bovine serumand 1% (v/v) of N3 supplement as previously described (Cardenas A. M. etal., Neuroreport, 1999, 10(2):363–369) and 1% (v/v) Site+3 supplement(SIGMA). Cells were allowed to differentiate for one week.

Cytology of RCSN-3 cell line. Cells were fixed in formaldehyde 4% inphosphate buffer pH 7.4. Cytochemical reactions included:Hematoxilin-eosin staining, ferrous ion capture to demonstrate thepresence of melanin in the form of neuromelanin,paraformaldehyde-glyoxylate staining to demonstrate the presence ofcatecholamines.

Immunohistochemistry of RCSN-3 cell line. Fixed cells were permeabilizedin an ascending/descending alcohol battery ranging from 50% to 96%. Theblocking reaction was carried out using BSA 1% in phosphate buffer. Theantibodies utilized were: (i) neuronal markers: NSE (pre-diluted,BIOGENEX) Synaptophysin (pre-diluted, BIOGENEX), MAP-2 (1:1000, SIGMA);(ii) glial markers: GFAP (pre-diluted, BIOGENEX), S-100 (pre-diluted,BIOGENEX); and (iii) functional markers: TH (1:1000-1:1500, SIGMA). Theincubation with the primary antibodies was carried out overnight and anABC detection kit (BIOGENEX) was used to develop the reaction andutilizing DAB as chromogen. Specific primary antibodies, fluoresceinlabeled secondary antibodies and tetanus toxin were used to evaluate thepresence of neurofilament 200 kD and tetanus toxin receptor.

Intracellular Ca²⁺ measurements. For intracellular Ca²⁺ measurements,the cells were replated onto 35 mm culture dishes. The variations ofintracellular Ca²⁺ were assessed by Ca²⁺-imaging techniques usingFluro-3. The cells were incubated at 37° C. for 40–60 minutes with theindicator. The dishes were visualized in an OLYMPUS BH2 microscopeequipped with epifluorescence (halogen lamp). The microscope wasconnected to a Cooled Extended Isis digital camera (PHOTONIC SCIENCE,Ltd, Robertsbridge, UK) connected to a dedicated PC equipped with anAXON DIGIDATA 2000 digitizing board (AXON Instruments, Foster City,Calif.). Images were acquired at 12 bit resolution and 1 Hz usingcustomized software AXON Imaging Workbench 2.1.80 (AXON). Thecompositions of the normal extracellular solutions were (in mM): 135 or145 NaCl, 5 KCl, 2 MgCl₂, 1.5 or 2.5 CaCl₂, 10 4-(2-hidroxyethil)piperazine-1-ethanesulfonic acid (HEPES)-NaOH, 10 Dextrose (pH=7.4).

Surgical procedures and behavioral testing. Four adult male Fisher 344rats (200–250 g) were lesioned by unilateral injection of6-hydroxydopamine bromide at two sites along the medial forebrainbundle. Assessment of apomorphine induced rotational behavior (i.p.injection of 5 mg apomorphine per kg body weight, National HealthService, Chile) was carried out visually twice, once per week, beforetransplantation. Only rats with more than 160 rotations every 30 minuteswere utilized after three times after transplantation (days 30, 55, and80). For transplant, confluent cultures were washed in PBS anddissociated with 1% trypsin. 500,000 cells in a volume of 4 μL wereimplanted through blunt Hamilton syringes and deposited at AP +1.0 mm,ML −2.5 mm, and V −4.7 mm (coordinates relative to bregma), toothbar setat −2.5. Rotational behavior was assessed visually every two weeks aftertransplantation.

Purification and characterization of UCHT1 proliferation factor. Atwo-stage approach was used for the isolation and characterization ofthe UCHT1 proliferation factor: (i) collection of media conditioned byUCHT1 cell line; and (ii) identification of the proliferation factor(i.e., transformation promoting factor(s)) associated with the UCHT1tumor cell line, and testing in bioassay. Culture media is composed ofDMEM/Ham F12 nutrient mixture (1:1) (SIGMA Chemical Co, St. Louis, Mo.Cat# D8900), supplemented with 1 g/l bicarbonate. To this basal media,the following modifications were made: complete media: Contains 87.5%basal media, 10% adult bovine serum and 2.5% fetal bovine serum; 2%Serum media: 98% basal media+2% fetal bovine serum; and cryopreservationmedia: 70% basal media, 20% fetal bovine serum and 10% DMSO. When not inuse, cells were stored in cryotubes with cryopreservation media, andkept in liquid nitrogen. Cells were thawed within 90 seconds in a waterbath at 37° C. Thawed cells were seeded onto culture dishes and fedcomplete medium. The dishes were kept at 37° C., 100% humidity and 10%CO₂, and media was renewed every 3 days. When the cells reachedconfluence, a passage was performed. The cells were then washed with PBSand detached by trypsinization (trypsin 0.1%) and resuspended bypipetting. The cells were centrifuged at 1000 r.p.m. for 10 minutes, andthe supernatant was discarded. Cells were then seeded onto new dishes ata 1/20 slit and fed complete medium.

Cryopreservation for purification and characterization of UCHT1proliferation factor. Media was aspirated from the culture dishes, andthe cells were washed with PBS and detached with trypsin 0.1%. Thesuspension was centrifuged at 1000 r.p.m. and the cell pellet wasresuspended in cryopreservation media, at a density of 1×10⁶ per ml. Thesuspensions were placed in cryotubes and frozen in a first stage to −86°C., at a rate of 1° C./minute. After 24 hours, the cryotubes weretransferred to liquid nitrogen for the final storage.

Collection and pretreatment of UCHT1 conditioned media for purificationand characterization of UCHT1 proliferation factor. UCHT1 cells werecultured in 15 cm diameter Petri dishes to confluence. At this time,conditioned media was collected and frozen at −20° C. Media was thawedand refrozen in 3 more cycles. Later, the media was centrifuged at 5000r.p.m. for approximately 20 minutes and the supernatant was filteredthrough 0.2 μm porosity nitrocellulose filters. Media with serum andbasal media without serum exposed to confluent UCHT1 cultures for 24hours were collected. For chromatography studies, desalinization andconcentration procedures were performed. Media samples were passedthrough PD-10 columns containing SEPHADEX G-25 M (PHARMACIA BIOTECH),where the sample was diluted by a factor of 1.4. The samples were thenconcentrated in CENTRICON molecular filter vials (AMICON) bycentrifuging in a SORVALL RC-28S refrigerated centrifuge (DUPONT) at4800 r.p.m. for 2.5 hours 100 μl of Tween 80 at 0.1% was added per vial.

Gel electrophoresis for purification and characterization of UCHT1proliferation factor. Polyacrilamide gels containing sodium dodecylsulfate (SDS, SDS-PAGE) were used. A total of 1.4 g SDS for the union of1 g protein was considered, to achieve an adequate charge-masscorrelation. The gels were run in a BIORAD electrophoresis chamber (MINIPROTEAN II), at 12.5% acrylamide and 10 columns per sample. Thesensitivity of this gel is 0.1 μg to 1.0 μg of protein per band dyedwith bright Coomassie blue and 2 ng to 10 ng in silver stains. Theresolution is 15 kDa–60 kDa (Bollag, D. et al. (1996) Protein Methods,2^(nd) Edition). Gels ran at 200V for 45 minutes, using an EPS 3500 XLElectrophoresis Power Supply, (PHARMACIA BIOTECH) or Power Pac 1000(BIORAD). Isoelectric focusing (IEF) allows separation of proteins bynet charge, as they migrate in a pH gradient generated by an electricalfield, indicating the isoelectric point of the proteins. The PhastSystem method was used, with a commercial gel Phast Gel 1% Agarosa IEFcontaining Pharmalyte 3 to 9. This gel was selected to its broadspectrum of pI. The focusing stage is carried out continuously, and thegels are later dyed with silver nitrate and later the dye is removed.The gel has no gradient, so the respective pI is visualized linearly.

Chromatography for purification and characterization of UCHT1proliferation factor. Hydrophobic interaction and ion exchange resinswere used. An FPLC liquid chromatography kit was used, commanded by asoftware FPLC DIRECTOR. Appropriate binding and deadsorption bufferswere used. Elution gradients, column volumes and flows were determinedfor operation. The information gathered corresponded to conductivity andabsorbance at 280 nm which determine chromatographic profiles. AnEXPRESS-ION, Exchanger D column (WHATMAN INTERNATIONAL) usingdiethylaminoethyl in a cellulose matrix, DEAE-cellulose. The adsorptioncapacity is 61 mg of protein per ml. The buffer used was Bis-Tris 20 mMpH 7.0 for linking, and the same buffer with NaCl 1 M was used forelution. For hydrophobic interaction chromatography, 1 ml columns ofphenyl-sepharose and Butyl-SEPHAROSE FAST FLOW (SIGMA) were used. Theunion buffer was Bis-Tris or Bis-Tris Propane 20 mM pH 7.0 with(NH₄)₂SO₄ 0.7M. To create the gradient, the same buffer lacking ammoniumsulfate is used (Andrews, B. A. et al., Bioseparations, 1996,6:303–313). The strategy contemplates high salt concentration and pHbetween 6.5–8.0. Since the proteins are not well characterized, a resincolumn substituted with phenyl groups was preferable.

Protein content in UCHT1 conditioned media. Protein was determined bythe Bradford method with modifications (Deutscher, M. P. [1990] “Guideto Protein Purification-Methods in enzymology” Academic Press, Inc.,182), using Coomassie Brilliant Blue G-250. BSA (SIGMA) was used forstandardization. An ULTRASPEC 3000 spectrophotometer (PHARMACIA BIOTECH)was used. Exceptionally, protein was determined by the bicinconinic acidmethod, using a Protein assay kit (PIERCE).

Bio Assay of UCHT1 conditioned media fractions. The selection of anadequate bioassay to detect transformation with the various fractions ofUCHT1 media collected was desired. The cell lines used were the KGFRcell line, the NRK 52E cell line, and the human nueroblastoma cell line.The KGFR cell line was derived from the mouse fibroblast 3T3 cell line,and transfected with the receptor for EGF. The cells grow attached tosurfaces, in media composed of DMEM/F12 (1:1) (SIGMA) supplemented with10% fetal bovine serum, and passaged with standard trypsinization. TheKGFR cell line was used to establish a soft agar and a liquid mediaassay to test fractions of the UCHT1 conditioned media. The NRK 52E cellline (ATCC:CRL-1571), derived from normal kidney epithelia of a rat(Rattus norvegicus). The NRK 52E cell line expresses receptors for EGFand multiplication stimulating activity (MSA), and grows attached tosurfaces. NRK 52E cells are not transformed and exhibit contactinhibition in culture, a fundamental property in assays oftransformation and malignancy. The cells grow in DMEM/F12 1:1 media(SIGMA) supplemented with 10% fetal bovine serum (FBS). Passages weredone with standard trypsinization. Split ratio 1:3–4, with mediarenewals twice a week. Human neuroblastoma cells derived from an explantof tissue were derived from a biopsy of a patient and subsequentlycultured. The cells grow adhered to substrate in DMEM/F12 1:1 media(SIGMA) supplemented with 10% FBS, 10% adult serum and NGF (CALBIOCHEM)10 ng/ml. They were later adapted to 10% adult serum, 7.5% FBS and 5ng/ml NGF.

Soft Agar Technique for purification and characterization of UCHT1proliferation factor. This technique correlates in 90% totransformation, and is faster and less costly than working with animalmodels such as the Nude mouse to detect tumorogenicity. Cells are grownin soft agar for a week, and colonies are the evidenced with dyes. Cellsgrowing on this environment due so independently of anchoring, whichcorrelated with a transformed phenotype. The protocol for the soft agartechnique is as follows: (1) dilute agar 5% 10 times in culture media,to final concentration: 0.5%; (2) add 0.7 ml agar 0.5% to 3.5 cmdiameter culture dishes (base); (3) mix 0.2 ml cell suspension (Approx.3×10⁴ cells/ml) per dish; (4) 0.7 ml agar 0.3% is placed over the baseagar, and the dishes are kept in the incubator for 1 week (37° C., 100%humidity, 10% CO₂); (5) after 1 week, cells are dyed withp-iodonitrotetrazolium 0.5 mg/ml; and (6) cells are incubated for 24hours incubation size and number of colonies are estimated.

Precipitation with acetone. Two methods were used for precipitation: (1)with acetone; and (2) with ammonium sulfate. For precipitation withacetone, media lacking serum and kept in UCHT1 conditioned medium for 24hours was used. Total protein content was assessed with the bicoconinicacid assay. The proceeding begins with the centrifugation of serum freeconditioned media at 5,000 rpm for 20 minutes, after which thesupernatant is filtered at 0.2 μm porosity. Acetone was later added at−20° C., and precipitate was collected by centrifugation at 10,000 rpmfor 30 minutes. The supernatant was collected and more acetone wasadded. The precipitate was resuspended in 1 ml cold sterile PBS. Asecond precipitation followed with excess acetone, using 0.1 ml of everyPBS fraction and 1.5 ml acetone.

Precipitation with Ammonium sulfate. Serum free, UCHT1 conditioned mediawas centrifuged at 5,000 rpm for 20 minutes, after which the supernatantwas filtered at 0.2 μm porosity. pH was maintained in the range of7.0–7.5 and temperature at 4° C. at all times. Ammonium sulfate wasadded and mixed for 30 minutes to equilibrate the solvent with protein.The precipitate was collected after centrifugation at 10,000 rpm for 30minutes. The supernatant was reutilized for further precipitations, andthe final precipitates were resuspended in 200 μl cold sterile buffer.

Adaptation to defined media, serum deprivation. Defined mediacorresponds to DMEM/F-12, enriched with the supplements listed in Table2, which are trophic for thyroid cells Scopes, R. [1988] “ProteinPurification—Principles and Practice” Springer-Verlag Inc., New York).UCHT1 cells were gradually adapted to defined media using a gradualdecrease of the serum content: 12.5% to 9.4%, 6.3%, 3.1% and finally1.25%. The conditions were kept for two passages in each serumconcentration.

TABLE 2 Media composition per ml Origin TSH 10 mU bovine Transferrin 5μg human Insulin 10 μg bovine Somatostatin 10 ng synthetic GlyHisLys 10ng synthetic Hydrocortisone 1.00E−08 M synthetic

UCHT1 cells were gradually adapted to defined media using a gradualdecrease of the serum content: 12.5% to 9.4%, 6.3%, 3.1% and finally1.25%. The conditions were maintained for two passages in each serumconcentration.

Culture media, solutions. All solutions were prepared in sterile,tridistilled and deionized water. Phosphate buffered saline (PBS) pH7.4, contained 8.0 g/l NaCl (136.9 mM); 0.2 g/l KCl (2.7 mM); 1.5 g/lNaHPO₄ (10.6 mM); 0.2 g/l KH₂PO₄ (1.5 mM); D solution, pH 7.4 contained8.0063 g/l NaCl (137 mM); 0.4026 g/l KCl (5.4 mM); 24.1 mg/l Na₂HPO₄(0.17 mM); 29.9 mg/l KH₂PO₄ (0.22 mM); 1.0899 g/l glucose (5.5 mM);2.0196 g/l (5.9 mM). Trypsin was diluted 0.1% w/v in PBS. Culture mediacontained DMEM/F-12 supplemented with 1 gr/l bicarbonate and 40 μg/mlgentamicyn if required (Laboratorio Chile, 80 μg/ampoule)

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Rat Thyroid Cell Line (UCHT1)

A clonal cell line derived from a functional and transplantable ratthyroid tumor was established in continuous monolayer culture by the useof enzymatic dissociation followed by an alternate culture-animalpassage procedure (Caviedes, R. and Stanbury J. B., Endocrinology, 1976,99:549–554). Autonomous and transplantable tumors were used as a cellsource for cultures (Matovinovic, J. et al., Cancer Res., 1970, 30:540;Matovinovic, J. et al., Cancer Res., 1971, 31:288). Tumors weredeveloped by implanting thyroid tissue from rats that had been fed on aniodine-deficient diet for 14–18 months into ¹³¹I-thyroidectomizedanimals on a similar diet. After approximately 9 months, the tumors weretransplanted to animals on a regular diet. Well-differentiatedfollicular and function tumors, which arose 6 months later (secondgeneration), were selected to establish serial cultures.

Cells from several tumors were introduced into monolayer culture throughenzymatic dispersion, followed by the alternate culture-animal passage(Buonassissi, B. et al., Proc. Natl. Acad. Sci., 1962, 48:1184). Thenutrient mixture Ham's F-10, supplemented with 15% horse serum and 2.5%fetal calf serum, was employed for the establishment of the cell line.As soon as the cells had adapted to these culture conditions, they weretransferred to Dulbecco and Freman's modified Eagle Essential mediumwith the same serum complement. Cultures were grown in Falcon plasticflasks in a humidified atmosphere (air containing 5% CO₂) at 37° C.Medium was renewed every 3 days, and the cells subcultured at intervalsof 8 days. At confluence, the cells from two Petri dishes were suspendedin 0.5 mL of isotonic saline solution and injected subcutaneously intothe thigh of each rat (2–3 months old). After approximately 2 months,the tumors were removed, dissociated by enzymatic exposure, and againplated in culture. A clonal line was isolated from one of the tumors,according to the single cell plating technique. Others were kept inprimary culture.

After being implanted back into the animal and again plated in culture,epithelial-like cells aggregated and rearranged themselves over thebottoms of dishes in structures resembling cross sections of a normalthyroid gland. The same morphology and growth pattern were maintainedafter innumerable subcultures and freeze/thawing periods. Cells grewwith a population-doubling time of about 24 hours in serum-supplementedsynthetic medium. Cell monolayers stained with periodic acid-Schiff(PAS) showed a uniformly epithelial-like morphology; their cytoplasmcontained abundant PAS-positive material that was resistant to enzymaticdigestion with amylase. Thin-layer chromatography of acid-butanol cellextracts in primary and clonal cultures, followed by a specific andsensitive staining method for iodine compounds, demonstrated thepresence of monoiodotyrosine (MIT), diiodotyrosine, andtriiodothyronine-thyroxine. In contrast, in similar extracts obtainedfrom a cultured liver cell line, the only iodinated amino acid was MIT.Thus, with regard to the above criteria, this cell line preservedspecialized thyroid cell morphology and function. Further detailsregarding materials and methods utilized, and observations as to culturemorphology and hormone detection, can be found in Caviedes, R. andStanbury J. B., (1976).

EXAMPLE 2 Preparation of UCHT1 Conditioned Medium (UCHT1-CM)

Glass Petri dishes (15 cm diameter) were inoculated with approximately5×10⁵ mycoplasma free UCHT1 cells (as described in Example 1) in a mixedsolution consisting of equal volumes of Ham F₁₂ and Dulbecco's modifiedEagle's medium (F₁₂/D) supplemented with 10% bovine serum, 2.5% fetalbovine serum, 0.015M HEPES (n-2-hydroxyethylpiperazine-N′-2 ethanesulfonic acid) buffer pH 7.2, 50 mg/i streptomycin sulphate and 100 mg/lsodium-penicillin-G, which was used as the basal growing medium (BGM).Cultures were incubated at 36° C., 100% humidity in an incubator withcontrolled 10% CO₂, 90% air atmosphere and total media changes everythree days. UCHT1-CM was collected from exponentially growing culturesand harvested from four subsequent culture periods of 24 hours,obtaining a total amount of 80 mL per dish at the end of the four days;the extensive cell detachment after confluence prevents further UCHT1-CMcollection. Finally, UCHT1-CM was filtered through 0.2 μm GelmanSUPOR-200 nitrocellulose membranes and stored frozen at −20° C.

EXAMPLE 3 Immortalized Skeletal Muscle Cell Line

A cell line (RCMH) in permanent culture was established from surgicallyremoved adult normal human skeletal muscle by exposure to conditionedmedia obtained from thyroid cells. Cells proliferated indefinitely butdisplayed density inhibition of growth while maintaining somedifferentiated markers. Under certain incubation conditions, cells fusedinto myotube-like structures, with a concomitant increase in musclespecific proteins, such as human myoglobin, skeletal muscle myosin,desmin and dystrophin, as identified using immunocytochemicalprocedures. In addition, RCMH cells displayed high affinity receptorsfor α-bungarotoxin (B_(max)=0.7 pmol/mg protein, K_(d)=1.5 nM) anddihydropyridines (B_(max)=0.3 pmol/mg protein, K_(d)=0.5 nM for[³H]PN200-110). These values are comparable to those reported for normalmuscle cells in primary culture. Patch-clamp studies showed the presenceof 42 pS carbachol gated channels and of 5 pS calcium channels (currentcarried by barium); chloride and potassium channels were also seen.Details regarding establishment of culture, culture conditions,immunocytochemical procedures, binding experiments, and single channelrecording carried out on the RCMH cell line, and results, are describedin Caviedes, R. et al., Biochimica et Biophysica Acta, 1992,1134:247–255.

EXAMPLE 4 Immortalized Cerebellar Cell Line

Ten Fisher, 6-month-old female rats were injected with 10⁶ UCHT1 cellssubcutaneously. After developing a tumor within 3 months, the animalswere anesthetized and portions of cerebellar cortex were dissected andplaced on watch glasses containing a mixture 1:1 of Eagles modifiedDulbecco's and Ham's F12 media (GRAND ISLAND BIOL. Co., NY, U.S.A.)without serum. Cerebellar explants of approximately 1 mm³ were prepared,placed in glass petri dishes and allowed to and grow in the same mediummixture containing 15% bovine, 2.5% fetal bovine serum, 0.015H M Hepesbuffer, pH 7.2, plus 50 mg/l streptomycin sulphate, 100 mg/lsodium-penicillin-G and sufficient glucose for a total amount of 6 g/l.Cultured explants were incubated at 36° C., 100% humidity. Clones wereisolated from cerebellar established cultures at the 25th passage andafter 15 months in vitro. One clone (UCHCC1) was maintained in cultureand studied while the others were frozen. The cerebellar cell lineUCHCC1 retained a neuronal-like morphology; the addition ofdimethylsulfoxide (DMSO) to the culture medium elicited a reproduciblemorphological “differentiation” event, characterized mainly by processextension. In “differentiated” cells, veratridine significantlyincreased the update of ²²Na. Such enhanced uptake was blocked bytetrodotoxin (TTX) with a half-maximal inhibitory concentration of 0.9nM. Binding of an [³H]ethylenediamine derivative of TTX ([³H]en-TTX) tothe microsomal fraction prepared from the same DMSO-treated cells,showed a single class of receptors with a maximal binding (B_(max)) of a173 fmol/mg protein and a K_(d) of 1.1 nM. Thyroid UCHT1 cells and“undifferentiated” (cultured without DMSO) cerebellar cells, did notshow significant effects of veratridine on ²²Na-uptake, or [³H]en-TTXbinding. The “differentiated” nerve-like properties, induced byappropriate environmental manipulation, demonstrate the usefulness ofcerebellar UCHCC1 cells as a model system for the developing centralneuron. Further details regarding establishment of culture, cultureconditions, sodium flux assays, binding assays with[³H]ethylenediamine-tetrodotoxin ([³H]en-TTX), morphological studiescarried out on the UCHCC1 cell line, and results, are described inCaviedes R. et al. Brain Res., 1986, 365:259–268.

EXAMPLE 5 Immortalized Myocardial Cell Line

A cell line (RCVC) in permanent culture was developed from adult ratventricular cells; transformation was attained by incubation withconditioned media from the UCHT1 rat thyroid cell line. Specifically,ventricular cavities were removed from the hearts of decapitated Fisher344 normal male rats and removed of fat and mesenchymal envelopes, andfinely minced. The myocardial explants of approximately 1 mm³ wereprepared, seeded onto 10 cm diameter glass dishes and allowed to attachand grow in BGM plus 20% UCHT1 conditioned medium (UCHT1-CM).Approximately 25% of ventricular explants attached, started an outgrowthin two weeks, and attained confluence in 40 days. Initial outgrowth weresplit by trypsinization and EDTA, and sorted out using the “selectiveserial passage” method. Three successive passages followed by thecorresponding preplating period of 24 hours were carried out to selectthe slowest attaching cells. Cultures were incubated for periods devoidof cysteine, glutamine, and sera to eliminate fibroblasts. Myoblastenriched cultures were subcultured by trypsinization and diluted 1:2 to1:10, depending on proliferative capacity. After 3 months of continuouspropagation in culture, UCHT1-CM was removed from BGM withoutsignificant effect on cell growth parameters.

Immortalized ventricular cells having a doubling time of 20 hours,contact inhibition of growth, and which display some muscle markers suchas a high glycogen content and positive immunoreaction for myoglobin,α-sarcomeric actin, α-actinin and desmin were obtained. A microsomalfraction from these cells was shown to bind ³H-nitrendipine with amaximal capacity of 295 fmol/mg protein and an equilibrium dissociationconstant of 0.7 nM. Nifedipine-sensitive ⁴⁵CA²⁺ influx was evident inpartially depolarized cells (40 mM K⁻ in the incubation medium). Anequivalent influx, induced by the calcium channel agonist BAYK-8644 andCGP-28392, was obtained in normally polarized cells.

Patch clamp studies show slow inward currents that can be completelyblocked by 5 μM nifedipine; cells were induced to furtherdifferentiation by culturing in a hormone supplemented medium for 30days. Under this condition, fast, inactivating inward currents and alarge outward current became apparent. After 40–60 days, the cellsexhibit La³⁻-sensitive fast and slow inactivating inward currents thatresemble T and L-type Ca²⁺ currents. Further details regardingestablishment of the RCVC cell line, culture conditions,immunocytochemical studies, ³H-Nitrendipine binding studies, ⁴⁵Ca²⁻ fluxexperiments, patch clamp methodologies, and results, are described inCaviedes, P. et al., Mol. Cell. Cardiol., 1993, 25:829–845.

EXAMPLE 6 Impaired Cell Lines as Models of Disease

Cells proliferated according to the methods of the subject invention canhave a naturally occurring or induced pathological defect, such that thecells provide an in vitro model of disease. Thus, mutant, diseased, orotherwise impaired cells can be proliferated for drug screening for thatparticular disease. For example, pathological tissue has beentransformed using UCHT1 conditioned medium, producing cell lines fromskeletal muscle of patients bearing Duchenne muscular dystrophy,pancreatic ducts of patients with cystic fibrosis, and from nervoustissue of a murine model of human Down syndrome and Alzheimer's disease.

Knowledge of neuronal dysfunction in Human trisomy 21 (Down's syndrome)is critical for understanding of the mechanisms that give rise tonervous system impairment. Cholinergic function is one of the mostcompromised in Alzheimer's disease and Down syndrome, two conditionsthat demonstrate similar pathologies (Caviedes, P. et al., Brain Res.,1990, 510:229–236) and altered choline transport. The establishment ofstable, in vitro models of the nervous system would provide an importanttool to rapidly and accurately address these problems. Therefore, a cellline proliferated according to the methods of the subject invention canbe obtained having similar neurotransmitter dysfunction mechanisms asthe originating tissues, and which would serve as a model to studypotential therapies and/or further alterations of the cell function.

A cell line (CTb) from a T16 trisomic mouse continuously cultured usingthe UCHT1 rat thyroid cell line has been established (Allen, D. D. etal., Euro. J. Neurosci., 2000, 12:3259–3264), which can be used as an invitro model for Down syndrome. Trisomic 16 and normal fetuses wereobtained by breeding double heterozygous (Rb 2H/RB 32 Lub) males withnormal C5B7BL females. Pregnant females were anesthetized and killedafter 12–16 days of gestation. The fetuses were placed in phosphatebuffered saline (PBS) and the trisomic fetuses were identified by theircharacteristic massive edema. Whole brains from trisomic fetuses wereremoved and meninges were withdrawn, and the cerebral cortex wascarefully dissected. The tissues were sliced and suspended in 3 mL ofPBS containing 0.12% (w/v) of trypsin (SIGMA) and incubated for 30minutes at 37° C. The trypsin reaction was stopped by adding an equalvolume of plating medium, consisting of DMEM/Ham F₁₂ nutrient mixture(1:1) (SIMA) modified to contain 6 g/l glucose, 10% bovine serum, 10%fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin (SIGMA).The suspension was centrifuged and the pellet was resuspended in 2 mLplating medium. The tissue was dissociated by passages through afire-polished Pasteure pipette, and the cells were then plated in acollagen-coated (CALBIOCHEM) culture dish at a density of 40,000/cm². Atthe time of seeding, the plating medium was supplemented with 10% (v/v)of UCHT1 conditioned medium. After 24 hours, the initial plating mediumwas replaced by feeding medium consisting of DMEM/Ham F₁₂ nutrientmixture (1:1) modified to contain 6 g/l glucose, 10% bovine serum, 2.5%fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10%UCHT1 conditioned medium. The cultures were kept in an incubator at 37°C. with 100% humidity and an atmosphere of 10% CO₂ and were monitoredroutinely for the appearance of transformation foci or morphologicalchanges, which became evident after variable periods of time (7–8months) and signaled the establishment of cell lines CNh (derived fromnormal cortex) and CTb (derived from trisomic cortex). Further detailsregarding the establishment and characterization of the CTb trisomiccell line are described below and in Allen, D. D. et al. (2000) andCardenas A. M. et al., Neuroreport, 1999, 10(2):363–369. Normal andtrisomy 16 spinal cord cell lines and dorsal root ganglion cell lineshave also been produced.

A human muscle cell line (RCDMD) was established from a Duchennemuscular dystrophy patient by culturing explants in the presence ofUCHT1 conditioned medium. The cell line has had over thirty passages andhas recently been cloned. The mother cell line is immunohistochemicallypositive for desmin, myoglobin, skeletal myosine, and a actinin, and isnegative for dystrophin. Further details regarding establishmentcharacterization of the RCDMD cell line are described in Caviedes, P. etal., Muscle & Nerve, 1994, 17:1021–1028 and Liberona, J. L. et al.,Muscle & Nerve, 1998, 21:902–909.

EXAMPLE 7 Miscellaneous Immortalized Cell Lines from Rat, Mouse, Bovine,and Human Sources

Two rat cell lines (RCHT-1 and RCHT-2) have been established byculturing explants from the hypothalamus of Fisher 344 rats in thepresence of UCHT1 conditioned medium. Markers detected byimmunohistochemistry in cell perikarya (% of positive cells) arepresented. Values are those obtained from non-differentiated cells. LHRH(+): 10%; tetanus toxin (+): 50–60%; neurophysin: 1%; ACTH: 1%; α MSH:1%; β endorphin: 1%; somatostatin: 1%; methenkephalin: 0.5%; TRH: 0.5%;vasopressin: 0.1%: oxytocin: 0.1%; tyrosine hydroxilase: 0.1%; GAD:0.1%; CRH (−); GFAP (−); S100 (−); NSE (−); N-epinephrine uptake:present; norepinephrine (by HPLC): >10 ng/mg protein; and dopamine (byHPLC): 13 ng/mg protein.

A rat atriocardiocyte cell line (RCAC) has been established by culturingexplants in the presence of UCHT1 conditioned medium.

Several cell lines have been established from the nervous system ofnormal and trisomy 16 fetal mice by culturing explants in the presenceof UCHT1 conditioned medium. As described above, the latter isconsidered an animal model of human trisomy 21 (Down Syndrome) andAlzheimer's disease. The murine cell lines originated from the cerebralcortex, hippocampus, spinal cord, and dorsal root ganglia of both normaland trisomic subjects. Cortical cell lines CNH (normal) and CTb(trisomic) immunohistochemically possess neuronal markers (NF, NSE,synaptophysin, MAP-2, etc.) and lack glial markers (GFAP, S-100). Thesemurine neural cell lines respond to glutamatergic (glutamate, NMDA,AMPA, and kainite) and cholinergic (nicotine) stimuli with increase inintracellular Ca²⁺. The CTb cell line expresses large intracellularvacuolized deposits of amyloid, evidenced by both Congo Red staining andimmunohistochemistry. Further details regarding the establishment andcharacterization of these immortalized murine cell lines are describedin Cardenas A. M. et al., (1999); Allen, D. D. et al. (2000).

A bovine corneal endothelial cell line has been established by culturingexplants in the presence of UCHT1 conditioned medium. The immortalizedcell line is immunohistochemically positive for Von Willebrand Factorand PECAM. The cells develop tube like structures when cultured onMATRIGEL.

A human ovarian granulose cell line was established by culturingexplants in the presence of UCHT1 conditioned medium. The immortalizedcells produce estrogen and progesterone at basal levels, and respond toFSH and LH with increments in the production of the former steroidalhormones.

A human thyroid cell line was established by culturing explants in thepresence of UCHT1 conditioned medium. The immortalized thyroid cellsproduce thyroglobulin and incorporate tritiated iodine.

EXAMPLE 8 Immortalized RCSN-3 Cell Line and Transplantation into RatStriatum

Primary cultures of the RCSN-3 cell line, derived from the substantianigra of an adult rat, were grown in the presence of UCHT1 conditionedmedia. The RCSN-3 cell line was grown on monolayers, with a doublingtime of 52 hours, a plating efficiency of 21% and a saturation densityof 410,000 cells/cm, when kept in feeding medium. FIGS. 2A–2F show thatundifferentiated RCSN cells tend to exhibit an epithelial likemorphology, with short or no processes and a more acidophylic cytoplasm.After differentiation, cell proliferation is greatly reduced, and theRCSN cells develop processes and establish contact with neighboringcells. The presence of melanin was evidenced with the ferrous ioncapture technique, demonstrating a homogenous distribution of thepigment in the cytoplasm, with faint labeling in undifferentiated stagesand a substantial increase upon differentiation.

Immunohistochemical characterization demonstrated that RCSN cellsexpress neuronal traits, evidenced by the positive immunolabelling forNSE, synaptophysin and MAP-2. NSE and synaptophysin show a fine granularpattern evenly distributed in the cytoplasm, as shown in FIGS. 3A–3H.Synaptophysin is especially intense at the zone of cell-cellinteraction. MAP-2 shows a fibrillary pattern of labeling, surroundingvacuole-like cytoplasmic structures. Neurofilament 200 kD labeleddifferentiated cells homogenously, and tetanus toxin is present in thecell membrane in a patch-like distribution. Functional neuronal markersare shown in FIGS. 4A and 4B, which present immunohistochemical stainingfor tyrosine hydroxylase. The labeling is slightly less intense innon-differentiated cells, and the label is distributed in the entirecytoplasm following a granular pattern. The presence of catecholaminesis also clear from the micrographs presented in FIGS. 4C and 4D, with acytoplasmic distribution. Glial markers GFAP and S100 were negative inboth control and differentiated conditions. When differentiated, up to40% of Fluo-3 loaded RCSN cells responded with increases in Ca²⁺ whenstimulated externally with 200 μm glutamate, and even more intenselywhen using simultaneous depolarizing conditions (70 mM K⁺), a situationdepicted in FIG. 5. Of 16 cells explored, the Ca²⁺ signal peaks afterone second of stimulation, and returns to basal level between 30–40seconds after the peak. These experiments show that the RCSN-3 clonalcell line retains general properties of neuronal tissue, and possessesspecific characteristics of the SN, such as the presence of tyrosinehydroxylase, DOPA decarboxylase, and catecholamines.

Suspensions of RCSN cells were transplanted into the striatum of ratswith 6-OH dopamine-induced lesions of the substantia nigra. FIGS. 6A and6B show typical patterns in the evolution of the rotational behaviorafter transplantation, one of which is characterized by a smooth,decreasing exponential-type curve, which levels off after 12 weeks atapproximately 25% of the initial rotation rate, as shown in FIG. 6A.Another pattern involves a greater drop in rotations 2 weeks aftertransplantation, followed by an increase and later a sustained decreasein the rate of rotation to again plateau after 12 weeks, as shown inFIG. 6B. At 16 weeks, the rats were sacrificed, and sections of thestriatum were taken and immunohistochemically strained with TH and DOPAdecaroxylase antibodies. As shown in FIGS. 7A–7C, cells stainingpositively for both markers are present in the striatum, showing intenselabeling and neurite formation.

As described above, the RCSN-3 cell line induces a sustained andprogressive reduction in the rotational behavior of 6-OHdopamine-lesioned rats (75% of the initial rotation values after 16weeks post implant). No previous in vitro differentiation agents wereutilized in these transplantation experiments. This may prove apractical asset, as the cells either have enough dopaminergic functionat the time of inoculation, or the in vivo microenvironment in thestriatum may be enough to sustain or induce a differentiated phenotypein the RCSN-3 line.

EXAMPLE 9 Immortalized RCSN-3 Cell Line in Hemiparkinson Model

Five Fisher 344 rats with weights between 180 and 200 g. were used(Group A: control (n=1), no lesion or transplant; Group B: controllesion (n=1), lesioned rat, no transplant; Group C: experimental group(n=3), lesioned and transplanted rats). Animals were fed similar adlibitum diets and water. Both transplant and lesion procedures were madeunder general anesthesia with ketamine and using a David Kopfstereotome. In order to induce the parkinsonian model, a estereotaxiclesion with 8 microg of 6-OHDA in 4 microl of saline solution wasinjected in the ventral tegmental area that contains the ascendingmesoestriatal dopaminergic pathway without jeopardizing the neuronalbodies. The coordinates were 4.4 mm AP, 1.2 mm lateral with respect tobregma and 7.8 mm vertical with respect to the surface of the brain. Theinjections were made with 50 microl Hamilton syringes. This lesiondenervates the areas with dopaminergic inervation of the ipsilateralstriatum (Urgenstedt and Herrera-Marschitz, 1981). The 6-OHDA causes thedeath of dopaminergic neurons of the substantia nigra and therefore theinterruption of the nigroestriatal pathway.

In the experimental group, after 8 weeks post lesion, a total of 6implants of RCSN-3 cell suspensions were made at different depths in thestriatum ipsilateral to the lesion, in order to restore thenigroestriatal circuit locally. After 6 weeks, the rats were sacrificedto carry out the morphologic evaluation. The rats were anesthetized withether and perfused via ventricule with a PBS solution for 10 minutesapproximately, to obtain a better fixation and clean the sample of redblood cells. Later, postfixation was carried out in two stages: First,the complete brain was fixed during 4 days, and a second fixation of thetissue sample to analyze (corresponding to the SN and striatum) for aminimum of 7 days. 25 microns thick frozen sections were made in acriostat at −20° C. The sections were collected in 0.05%sodium azide inPBS.

The immunoenzymatic technique used was the detection of the enzymetyrosine-hydroxilase. The sections obtained were washed with PBS and theblockade of endogenous peroxidase was performed with hydrogen peroxidediluted to 3% in PBS at saturating concentrations. Washing with PBS wasrepeated twice and the block of non-specific labeling was carried outwith BSA 1%, sodium azide 0.05%, Triton X100 1% in PBS during 60minutes. Sections were then incubated without washing in primaryantibody 1:2500 (MONOCLONAL ANTI-TYROSINE HYDROXYLASE, CLONE TH-2) at 4°C. for 12 hours. The sections were then washed with BSA 1% in PBS twice(all the subsequent washes were made with the same solution) andincubated with secondary antibody 1:100 (ANTI-MOUSE INMUNOGLOBULINS,Biotin conjugate) for 60 minutes. Three 10 min washes were made andthree sections were incubated with the Avidine-HRP complex 1:100(EXTRAVIDIN Peroxidase conjugate 0.5 mg/ml) for 60 minutes and a secondsequence of three washes of 10 minutes each was performed. The sampleswere incubated in chromogen DAB (3.3 diaminobenzidine), for 3 to 5minutes, were washed twice, mounted and covered for later analysis atthe microscope.

The analysis of the histological sections positive for TH shows thecerebral areas that express this enzyme with a strong brown color labelin the cytoplasm and axonic terminals of TH⁺ neurons (FIG. 8). In thesections, certain areas with a tenuous positive coloration due to thepresence of certain degree of background can be appreciated, due toproteins that bind to the antibody non specifically, that are notcomparable with the TH-positive areas where a strong labelling isevident in the zone of implants.

When observing sections of the normal rats (control group A, FIG. 8) aclear symmetry in the labelling is observed, with labelling in striatalareas. When compared with the sections of the lesioned rats (lesioncontrol group of B, FIG. 9) they display a noticeable asymmetry in thelabelling pattern, lacking the characteristic TH⁺ labelling in the 6 OHdopamine-lesioned side, reflecting the absence of dopaminergicterminals.

At 4× magnification, in the sections of the experimental group (FIG.10), a TH⁺ localized region is evident, specially when comparing withthe rest of the striatum. When analyzing the lesioned area, small zonesof dark brown color are evident, in special in proximity to the lateralventricles, that correspond to the viable transplanted cells.

At greater magnification (20×) (FIG. 11) in this zone, an accumulationof TH+ structures can be observed in the zone of implants, associatedwith the needle tract and near the ventricles, where theoretically therestoration of dopaminergic interaction with striatum is most necessary.

Through further magnification (40×; 100×) and with greater detail (FIGS.12, 13, and 14) it is possible to observe that these TH⁺ zonescorrespond to cells with a morphology that exhibits a body and processesof a neuronal type. These processes, which present some ramifications,follow a direction towards the striatum. In addition, these cells arenot isolated, they appear in small groups, associated to the zones ofgreater labelling (where it is not possible to recognize individualcells, due to high cell density and strong labelling).

Positive TH labelling confirms the existence of dopaminergic neurons indifferent areas from the brain, since such cells express within theirenzymatic machinery this rate limiting enzyme that converts of tyrosineinto L-Dopa, which in turn undergoes decarboxilation to become dopamine(Adams, R. et al. (1999) Principios de Neurologia. Editorial McGraw-HillInteramericana, México DC, 6th Edition, pp. 925–931). In addition,according to our results, a strong TH⁺ reaction is observed in zonesrich in dopaminergic terminals such as the substantia nigra andstriatum, which confirms that TH⁺ neurons (terminals) are indeed presentgiven their morphologic location, since the nigroestriatal pathwaye actsmainly as a modulator of the basal ganglia by dopamine (Kandel, E. etal., (2000) Principles of Neural Science, Editorial McGraw-Hill, USA,4th edition, Chapter 15, Neurotransmitters, pp. 280–297).

The sections studied in control rats show symmetry in labelling betweenboth striatum, since they have both nigroestriatal pathways intact.Conversely, lesioned injured rats display a unilateral neuronaldegeneration of the pathway, losing all connections between thesubstantia nigra and the striatum, which is confirmed in the sectionscuts of the lesioned rats.

In this study, the presence of implanted dopaminergic RCSN-3 neurons wascomfirmed, as evidenced by the presence of discrete regions of TH⁺ cellsin the striatum the lesioned side limited to the lateral wall of thelateral ventricle, and which present the same morphology that thoseneurons that are adhacent to the space left by the veedle tract.

The present work is the result of the experience of implants ofimmortalized cells of adult substantia nigra in an animalhemiparkinsonian model (Cenci, M.A. et al, Nature Reviews Neuroscience,July 2002, 3(7):574–9), that allows us to envision a possibledevelopment of definitive therapies for Parkinson's disease, which canreplace present pharmacological therapies (Rascol, O., J. Neurology,April 2000, 247(Suppl. 2):II 51-7; Weiner, W., Archives of Neurology,March 2000, 57:408). The model of implants of immortalized cellspresents advantages over other experimental and applied techniques inmedicine for the surgical treatment of the disease. Firstly, theimmortalized cells derived from dedifferentiated adult tissue which doesnot involve ethical conflicts like transplants of embryonic cells(Jong-Hoon, K. et al., Nature, Jul. 4, 2002, 418:50–56; Björklund, L.,et al., PNAS, February 2002, 99:2344–2349; Freed, C., PNAS, February2002, 99:1755–1757), fetal (Blanco, L. et al., Reviews Neurology, March1998, 26(151):361–5; Vogel, G., Science, March 2001, 291:2060–2061), orfrom stem cells (McKay, R., Nature, Jul. 27, 2000, 406:361–364; McLaren, A., Nature, Nov. 1, 2001, 414:129–131), implied in the use ofembryos. Secondly, cells immortalized from the host would not requireimmunosuppression as the present treatments (Lindvall, O. and P. Hagell,Clin. Chem. Lab. Med., April 2001, 39(4):356–61; Dunnett, S. et al.,Nature Reviews Neuroscience, 2002, 2:365–69; Jankovic, J., Archives ofNeurology, July 1999, 56:785; Fischbach, G. and G. McKhann, N. Engl. J.Med, March 2001, 344:763–765), therefore diminishing the risksassociated with this type of therapeutic approach.

In summary, with the present study, the presence of RCSN3 neurons in thestriatum of transplanted rats is confirmed, as evidenced by TH⁺staining. In addition, the presence of cytoplasmic processes could bethe morphologic base of the reestablishment of the synaptic connectionswith the interneurons of the striatum that would explain the improvementin the rotational conduct induced by apomofina in hemiparkinsonianomodel of rat.

EXAMPLE 10 Amperimetric Detection of Dopamine Secretion in RCSN-3 Cells

Electrochemical detection of catecholamine release was performed asdescribed previously (Kawagoe, K. T. et al., Analytical Chemistry, 1991,63:1589–1594). Briefly, carbon fiber sensors were constructed byinserting single carbon fibers (of 10 μm diameter) into pulled glasscapillaries. The carbon fiber electrode was then coated with a thin anduniform isolation film using the technique of anodic electrophoreticdeposition of paint. The polymer film was then heat cured. Before eachexperiment, the electrodes were polished at a 45 degree angle on amicropipette beveling wheel (NARISHIGE, Tokyo, Japan). Electrochemicalcurrents were amplified with a List EPC-5 patch-clamp amplifier. Thepotential of the carbon fiber electrode was set at +650 mV. The currentsignal was filtered at 10 kHz through a low pass filter, stored, andanalyzed with an IBM PC-compatible computer.

Cells were cultured in 1 cm diameter coverslips using either growthmedia (F 10 supplemented in 10% adult bovine serum, 2.5% fetal bovineserum, 100 U/ml penicillin and 100 μg/ml), or differentiation media,where adult serum was reduced to 2% and fetal serum was omitted.

To verify if RCSN-3 cells can produce MPP⁺ from MPTP, lisates of RCSN-3cells (differentiated and non-differentiated) were incubated with MPTP.Differentiation was induced by culturing in F10 media+2% adult bovineserum for one week. Non-differentiated conditions were attained with F10media+10% adult bovine serum and 2.5% fetal bovine serum. Lisate bufferwas PBS with 50 μM PMSF and a protease inhibiting cocktail (leupeptin,pepstatine, chemostatin). Samples were incubated with MPTP and MPP⁺ for15 min. at 37° C. HPLC studies were carried out in a SHIMADZU HPLC withreverse osmosis, reading at 245 nm. Results are shown in FIGS. 16A–C,FIGS. 17A–D, and FIGS. 18A–C. FIGS. 18A–C demonstrate no production ofMPP⁺ (absence of peak at 14.4 min.) when cell extracts are incubatedwith MPTP, suggesting that MAO B activity is not present in these cells.

EXPERIMENT 11 Apoptosis in the RCSN-3 Cell Line

FIGS. 19A and 19B show DNA fragmentation studies (TUNEL) in the RCSN-3cell line cultured in control conditions (FIG. 19A) and after treatmentwith MPP+ (FIG. 19B). Note the fragmentation of DNA in MPP+ treatedcells, indicating an apoptotic mechanism is involved. FIGS. 20A–20C showmitochondrial membrane potential changes with the JC-1 dye. JC-1 is anovel cationic carbocyanine dye that accumulates in mitochondria. Thedye exists as a monomer at low concentrations and yields greenfluorescence, similar to fluorescein. At higher concentrations, the dyeforms J-aggregates that exhibit a broad excitation spectrum and anemission maximum at ˜590 nm. These characteristics make JC-1 a sensitivemarker for mitochondrial membrane potential (MOLECULAR PROBES, Eugene,Oreg.). The lower flourescence in dopamine and Mn treated cellsindicates a decrease in membrane potential, suggesting thatmitochondrial dysfunction underlies the effect of these substances.FIGS. 21A and 21B also show JC-1 staining in the presence of MPP+,suggesting that this toxin also affects mitochondrial function. FIGS.22A and 22B show the ratio of emission between JC-1 and J monomer,normalized according to basal fluorescence. Dopamine, manganese and MPP+exhibit significant differences from controls.

EXAMPLE 12 Oxidation of Melanin in RCSN-3 Cells

RCSN-3 cells were preincubated for 24 hrs in the presence of 100 microML-Dopa. Cells were lysed with a hypotonic buffer. Melanin oxidized insitu exhibited a lipofuscin-like yellow fluorescence. Oxidation ofmelanin in vitro degraded the melanin polymer, resulting in afluorescent solution. Fluorescence spectroscopy gave an excitationmaximum at approximately 470 nm and an emission maximum at approximately540 nm for both natural and synthetic melanin. Increasing the time ofexposure to light or hydrogen peroxide increased melanin fluorescence(Kayatz, P et al., Invest Ophthalmol Vis Sci, January 2001,42(1):241–6).

TABLE 3 Average STD Plastic L-DOPA (−) 0.010 0.007 Plastic L-DOPA (+)0.040 0.012 Glass L-DOPA (−) 0.012 0.001 Glass L-DOPA (+) 0.033 0.003

Tables 4 and 5 show a one-way analysis of variance (ANOVA) summary table(Table 9) and a Student's t-Test summary table (Table 10).

TABLE 4 Group 1 Group 2 Group 3 Group 4 d name Plastic, Plastic, Glass,Glass, Control L-DOPA Control L-DOPA n 2 3 3 3 mean 0.0103347450.040450776 0.011709213 0.03305022 SD 0.006677779 0.0123016690.001290085 0.003228081 1-way ANOVA F = 11.7508 p = 0.004021309 Rejectthat groups have no differences (thus, at least one pair of groups willreach statistically significant difference)

TABLE 5 Results of Student's T Test T value of the Student's t-tests forunpaired observations P value Plastic, Plastic, Glass, Glass, 2-tailedNames Control L-DOPA Control L-DOPA Plastic, −3.06637 −0.37672 −5.32810Control Plastic, 0.037425 4.02468 1.00786 L-DOPA Glass, 0.7255120.015803 −10.63299 Control Glass, 0.005973 0.370539 0.000443 L-DOPA

EXAMPLE 13 Pancreatic Cell Line

A sample of newborn Cebus monkey pancreas was obtained and digestedenzymatically with collagenase 0.2%. Cells have undergone six passagesand most cells express insulin content (80%). Approximately 20% expressglucagon. This proportion could be due to selection due to the highcontent of glucose of our standard DME/HamF12 media (3.15 g/l) used inthe UCHT1 protocol. However, the sample was taken from the tail, whichhas a higher proportion of insulin containing cells. Established fromthe tail of a pancreas of a newborn Cebus monkey. The cell line hasundergone 4–5 passages, with over 20 population doublings. The cellslook epithelial in morphology. Immunofluorescent studies show over 90%positive reaction for insulin, and less than 5% for glucagon, in culturemedia with 3.15 g/l glucose. These cultures will continue to beproliferated until immortalization is evident and cultures becomeindependent of UCHT1 media, at which point a full characterization willbe carried out.

EXAMPLE 14 Immortalized Neural Cell Lines and Implantation for Treatmentof Neurological Disorders

Serotonergic cell lines can be derived from the lateral hypothalamus,dorsal raphe nucleus, or hindbrain of embryonic, neonatal, or adultorigins. Such cells can be transplanted for the treatment of pain andspinal cord injuries, among other neurologic disorders.

A noradrenergic cell line can also be obtained, derived from the locusceruleus, the nucleus interstialis striae terminalis, the dorsomedialnucleus, or the raphe nucleus from embryonic, neonatal, or adult donors.Such a cell line can be used for the treatment of epilepsy or memorydisorders, for example.

Purkinje cells of the cerebellum can be proliferated from fetal,neonatal, or adult sources for transplantation in cerebellar ataxiadisorders, including hereditary or sporadic cerebellar ataxias, familialcerebello-olivary atrophy, ischemia affecting the cerebellum,ataxia-telangiectsia, or immunopathological paraneoplastic degeneration,as well as other forms of ataxia.

Another neuronal cell line of the subject invention includes spinal cordor brain stem motor neurons, which can be proliferated from embryonic,neonatal, or adult sources for transplantation for the treatment ofamyotrophic lateral sclerosis or following peripheral nerve injury.

Glial cells from other sites, such as cortical oligodendrocytes,oligodendrocyte progenitor cells, glial neural stem cells, as well ascortical or other glial cells, and peripheral Schwann cells can beproliferated for treatment of multiple sclerosis, peripheral nerveinjury, other demyelinating disorders, as well as following head trauma,spinal trauma, hypoxia, ischemic brain disorders/stroke, or opticneuropathy. Such cells can be derived from stem cells, precursor orprogenitor cells, or mature cells, from embryonic, neonatal, or adultsources.

Using the methods of the subject invention, striatal, as well asmesencephalic, glial cell lines can be established. Such cell lines willbe useful for numerous therapies, including co-grafting with dopamineneurons, co-grafting with striatal neurons, transplantation adjacent,rather than mixed in with, other transplants in order to encourageelongation or branching of neurites, as desired for the particulartransplant paradigm. Alternatively, such cell lines can be used as astand-alone therapy, e.g., to slow or reverse the progression ofdegenerative disorders affecting the substantia nigra or the striatum,as in Parkinson's disease or Huntington's disease. Such cells are usefulfor transplantation purposes following a stroke that involves thestriatum in other brain regions. Likewise, glial cells can participatein providing trophic support for nigral or striatal neurons, and mayameliorate the neurodegeneration seen in Parkinson's disease orHuntington's disease. These glial cells can be useful in preventingtoxin-induced neurodegeneration or neurotoxicity, and provideneuroprotection or rescue of damaged nigral or striatal neurons. Thesecells or other glial cells can be used in treating demyelinatingdisorders.

Establishment of a mesencephalic glial cell line can be achieved byobtaining glial cells from the fetal, neonatal, adolescent, or adultsubstantia nigra (mesencephalon). The striatal cell line includes glialcells derived from the fetal, neonatal, adolescent, or adult striatum.Such cell lines are created by exposing or contacting primary tissuederived from these sites to proliferative factor from the UCHT1 ratthyroid cell line, as previously described. The UCHT1 proliferativefactor can be isolated or contained within a composition, such asconditioned medium from the UCHT1 cell line. Human dopamine cell linesderived from the fetal mesencephalon, if used, is preferably dissectedfrom Carnegie Stage 18–23 donors (Freeman T. B. and Kordower J. H.,Human cadaver embryonic substantia nigra grafts: effects of ontogeny,preoperative graft preparation and tissue storage, in IntracerebralTransplantation in Movement Disorders: Experimental Basis and ClinicalExperience, 1991, Elsevier Science Publishers, Amsterdam 15:163–169;Freeman T. B. et al., Exp. Neurol., 1991, 113:344–353; Freeman T. B. etal, Annals of Neurol., 1995, 38:379–388). The ontogeny of glial cellsalso occurs within this window, but these cells also have a broaderontogeny window than the dopamine neurons that develop in a restrictedtime frame.

Nigral tissue is exposed to the proliferation factor from the UCHT1 ratthyroid cell line, as described above, wherein after about 1–8 months ofexposure, cells enter a permanently dividing but differentiated state.Several different cell lines are derived from the mesencephalon,including dopaminergic cell lines as well as glial cell lines.Immunohistochemical markers of neuronal differentiation, such astyrosine hydroxylase, and glial differentiation, such as GFAP, will beused. Furthermore, in vitro tissue culture methods are utilized todemonstrate that such a cell line induces neuritic outgrowth in dopamineneurons in vitro for the step in characterization, among others.

A striatal glial cell line can be established using fetal, neonatal,adolescent, or adult striatal tissue dissected using known methods(Freeman T. B. et al., Cell Transplant, 1995, 4:539–545; Freeman T. B.et al., Human fetal tissue transplantation for the treatment of movementdisorders, in Neurosurgical Treatment of Movement Disorders, AANSPublications, New York, N.Y., pages 177–192; Freeman T. B. and BorlonganC. V., Soc. Neurosci. Abst., 2000, 26:209.6; Freeman, T. B. et al.,Proc. Nt. Acad. Sci., 2000, 97:13877–13882). Similar tissue culturemethods for the creation of the cell line and characterization of theglial component can be performed. It is anticipated that GABAergic,cholinergic, and other cell types found within the striatum would befound within the culture, and such cell lines can also be characterizedimmunohistochemically, in vitro, and following transplantation, asabove.

Another aspect of the invention is directed to a method for producing acell line useful for transplantation purposes in patients withParkinson's disease, comprising the steps of dissecting cells from thehuman neonatal, adolescent, or adult substantia nigra or the human fetalmesencephalon at Carnegie Stages 18–23. During these stages ofdevelopment, dopamine neurons develop in the embryonic nigra, and graftsfrom this stage survive transplantation, form neuritic extensions, andconnect to the rodent and human brain, inducing behavioral and clinicalbenefit, respectively. Success of the use of this donor age has beendemonstrated clinically (Freeman, T B et al. Ann. Neurol., 1995,38:379–388), as well as at autopsy, where grafts were found to survive18 months after transplantation (Kordower, J H et al., N England J Med,1995, 332:1118–1124; Kordower, J H et al., J Comp Neurol, 1996,370:203–230; Kordower, J H et al., Cell Transplant, 1997, 6:213–219).

The nigral tissue or human fetal mesencephalon, which contains 10%dopaminergic nigral neurons, is exposed to the UCHT1 proliferationfactor, for example, by exposure to UCHT1 conditioned medium. Thisprocedure has been performed using rodent nigral tissue. The cell linescreated in this way differ from cells known prior art in that: (1) Thecells are grown in vitro rather than derived directly from a humanfetus; (2) the cell line comprises exclusively dopamine neurons ofnigral origin; (3) cells are able to be produced using goodmanufacturing practices in a reproducible and reliable way; (4) thecells can be cryopreserved with preservation of adequate viability invitro as well as in vivo; and (5) the cells are available electively forsurgery.

As mesencephalic dopamine neurons constitute about 10% of the totalcells found in the ventral mesencephalon, numerous types of cell linescan be derived from this region, including dopamine neurons from thesubstantia nigra, which normally project to the striatum; dopamineneurons from the ventral tegmental area, which normally have mesolimbicand frontal projections; as well as mesencaphalic glia and otherneuronal types of cells. Dopamine neurons can be classified usingtyrosine hydroxylase immunohistochemistry, as well as markers fortyrosine hydroxylase RNA. Specific markers of nigral dopamine neurons,as opposed to those from the ventral tegmental area, may be used, suchas aldehyde dehydrogenase immunohistochemistry. In addition, as neuriticoutgrowth of nigral grafts is seen within the striatum, as opposed todopamine neurons from the VTA, where striatal outgrowth is not seen,confirmation of appropriate cell-cell interactions can be tested in vivowith transplantation of specific dopaminergic cell lines in a 6-OHDA ratmodel, where appropriate neuritic outgrowth within the striatum isexpected to be observed (Schultzberg, M. et al., Neurosci, 1984,12:17–32).

Thus, this method, as opposed to other methods known in the art, useshuman nigral tissue as the starting tissue, rather than rodent nigraltissue, permitting the creation of an allogenic cell line that can beused clinically without a need for immunosuppression. Also,graft-derived neuritic outgrowth from a human-derived cell line isanticipated to be significantly greater than that from a rodent orporcine cell line.

Another aspect of the invention is a method for creating a GABAergic(gamma-aminobutyric acid-producing) proliferated cell line that istransplantable in multiple patients having Huntington's disease, andalso for creating such a cell line for use in other diseases wheretransplantation of GABAergic cells is useful, such as in the treatmentof Parkinson's disease, epilepsy, schizophrenia, spinal cord injury,stroke, or other neurodegenerative diseases (Winkler, C. et al.,Experimental Neurology, 1999, 155:165–186).

In Parkinson's disease, transplantation of GABAergic neurons into thesubthalamic nucleus is beneficial in inhibiting output from this nucleusand ameliorates some symptoms or other movement disorders. In the caseof epilepsy, transplants around a seizure focus, or in other regions ofthe eleptogenic pathway suppress, seizure activity. In the case ofschizophrenia, GABAergic inhibition of the ventral tegmental areadopaminergic projections down-regulates the dopamine input to thefrontal and mesolimbic cortex and diminishes the symptoms. Following astroke involving the striatum, striatal transplants can provide clinicalbenefit, as has been demonstrated in similar animal models.

Stable human GABAergic neuronal lines that retain differentiatedproperties in continuous culture can be constructed by exposing humanneonatal, adolescent, adult, or fetal striatum to the UCHT1proliferation factor, e.g., via UCHT1 conditioned medium, as taughtherein. For example, human adult striatum or far lateral ventriculareminence tissue can be exposed to UCHT1 conditioned medium. Fetal tissueis preferably derived from a human embryo with a donor stage ofapproximately 7.5–9 weeks post-conception.

After exposure, stable, proliferating differentiated cell lines arederived from the cell types found in the far lateral ventriculareminence. GABAergic and cholinergic cell types, among others found inthe developing striatum, can be found within the different clones in theculture. Cell lines of each type of neuronal phenotype found can becreated. Cells can be characterized using immunohistochemical methodsknown in the art. Two different striatal GABAergic cell lines can becreated, one that co-labels with CCK and one that co-labels withenkaphalin (ENK), representing two different classes of GABAergicprojection neurons found in the striatum (Freeman, T. B. et al. Proc.Nat. Acad. Sci., 2000, 97:13877–13882).

Following the creation of the cell lines, the transformed cells can becharacterized immunohistochemically and transplanted into a Huntington'sdisease model in rats. If successful, such a cell line is then usedclinically for transplantation purposes, and may be mixed with otherstriatal cell types, such as cholinergic interneurons or striatal glia,among others. Such a cell line can also be used to explore thetherapeutic benefit in models of Parkinson's disease, epilepsy, spinalcord injury, stroke, and schizophrenia.

Another aspect of the invention includes methods for producing a cellline of striatal cholinergic interneurons for co-transplantationpurposes with a similar GABAergic cell line. Here human neonatal,adolescent, or adult striatal neurons, or human fetal cells derived fromthe medial ventricular eminence (Carnegie Stages 18–23) or the farlateral ventricular eminence (Carnegie Stages 23–29) after migration ofcholinergic progenitors to this region occur, are exposed to the UCHT1proliferation factor, as taught herein. It has been demonstrated thatthe UCHT1 conditioned medium, for example, can transform primaryparenchymal cultures into a substantially permanently dividingdifferentiated cell line. Such a human cell line is able to betransplanted without the need for lifelong immunosuppression, since thetransplant represents an allograft, from the immunologic perspective.

Another aspect of the invention involves the amelioration of memorydisorders associated with loss of cholinergic input in the human brainby transplantation of cholinergic neurons. Such neurons can be suppliedfrom cell lines derived from either the nucleus basalis or theseptum/diagonal band pathway as being the most likely to provideappropriate reinnervation of the brain in memory disorders.

Cholinergic neurons derived from the human septum and nucleus basaliscan be dissected from neonatal, adolescent, or adult brains or a humanembryo using known techniques. As previously discussed, the tissue isexposed to the UCHT1 proliferation factor, and the cell line created fortransplantation purposes. The cells can be characterized usingimmunohistochemical markers of cholinergic neurons, includingcholinacetyl transferase and acytyl cholinacetyl transferase, amongothers.

EXAMPLE 15 Sertoli Cell Lines and Other Cells Providing Immunoprivilege

Sertoli cells can also be proliferated using the methods of the subjectinvention. Sertoli cells can be dissected from any of a variety ofmammals (e.g., rodent, pig, human). Preferably, the Sertoli cells aredissected from the testicles in the prepubescent stage of the donor.During this stage, Sertoli cells provide maximum trophic support, aswell as expression of Fas-L, for example. Non-proliferated Sertoli cellshave been found to survive, provide neurotrophic effects on the brain,neurotrophic support of co-grafts, as well as provide localimmunoprotection for neural xenografts via Fas-L or other mechanisms, aswell as systemic allo- and xenografts (Sanberg, P. R. et al.,Transplant. Proc., 1997, 29:1926–1928; Willing, A. E. et al., BrainRes., 1999, 246–250; Willing, A. E. et al., Brain Res. Bull., 1999,48(4):441–444); Kin, T. et al., Cell Transplantation, 2002, 11:547–552).

As previously described with respect to all cells, Sertoli cells can beexposed to the UCHT1 cell line's proliferation factor (e.g., byculturing in UCHT1 conditioned medium) for a duration of about 1 toabout 8 months, until cells are transformed into a continuously dividingbut differentiated state. Thus, Sertoli cells can be proliferated, usingthe methods of the subject invention, for their FasL expression andco-transplanted to produce local immunosuppression without the need forsystemic immunosuppression.

Sertoli cells proliferated using the methods of the subject inventionhave several advantages, including: (1) the cells are able to beproliferated in vitro, allowing for a generation of adequate quantitiesof Sertoli cells for a variety of uses (e.g., manufacture ofbiomolecules, therapeutic implantation, and biological response models),as the availability of donor Sertoli cells is normally limited; (2) ahuman Sertoli cell line can facilitate xenograft or allograft co-graftsurvival, minimizing antigenic stimulation of the recipient; (3) thecell line can consist exclusively of Sertoli cells, and therefore couldnot be contaminated by other mesenchymal cells that may contaminatefresh batches of Sertoli cells; and (4) the cell line can bemanufactured using good manufacturing practices and cells can be storedusing cryopreservation for use electively.

Furthermore, ovarian stromal cells can be utilized in the subjectinvention to provide the same benefits as Sertoli cells (e.g.,immunosuppressive or trophic properties). For example, cografting ofislet cells with placental tissue can normalize blood glucose indiabetic mice (Suzuki, K. et al., Cell Transplantation, 2002,11:45–457), thus preventing rejection in a similar fashion to Sertolicells. Therefore, proliferation of placental tissue and ovarian stromalcells can be carried out according to the methods of the subjectinvention. Cells can be genetically modified to express genes encodingapoptotic products, such as those produced by Sertoli cells, ovarianstromal cells, and placental cells.

Lumbar disc material is relatively immune-privileged due to minimalblood supply found in the disc, as well as expression of Fas ligand.Therefore, disc material can be proliferated using the methods of thesubject invention and administered to patients with degenerative discdisease with or without immunosuppression, or with short-termimmunosuppression. Proliferated disc cells can be transplanted followingdiscectomy or trauma involving the disc. The cells can be administeredvia an open procedure or percutaneously under radiologic control (i.e.,fluoroscopy), for example.

EXAMPLE 16 Purification and Characterization of UCHT1 ProliferationFactor

The UCHT1 proliferation factor that causes immortalization of cell lineshas been partially characterized. The proliferation factor, a putativeglycoprotein, differs from the many transforming growth factors (TGFs)derived from conditioned media of normal and neoplastic cells, in thatknown TGFs induce a proliferating effect which is reversible uponremoval of the conditioned media; the effect of the UCHT1 proliferationfactor is long lasting, even after the medium is withdrawn. Studies inlaboratory animals clearly demonstrate the influence of thyroid hormoneson induction and growth of several types of experimental tumors (i.e.,lymphoma, mammary tumors, primary and transplanted hepatomas).Triiodothyronine plays a role in neoplastic transformation of culturedcells by X rays, chemical carcinogens, and DNA and RNA viruses. The factthat the cellular counterpart of a viral oncogene (v-erbA) encodes athyroid hormone receptor suggests that this hormone has either a director indirect effect on tumor growth. Nevertheless, the presence of thehormone by itself seems insufficient to induce transformation since nocell transformation or tumor promotion has been documented in responseto the sole effect of the hormone, and therefore the simultaneouspresence of known and unknown growth factors is required.

As described herein, the UCHT1 cell line has been shown to release oneor more factors (e.g., in culture media) that are capable of inducingproliferation and later immortalization in primary cell cultures, withpreservation of hystotypic and functional properties. Conditioned mediaderived from cell lines established by the UCHT1 conditioned media(e.g., RCMH, RCVC, and UCHCC1), and other clonal cell lines (e.g.,NIH3T3, PTK2, MDCK and KFR) do not show proliferating or transformingactivities when added to primary cultures.

Crude UCHT1 conditioned media, ultra-centrifuged at high supernatantspeed (HSS) at 100,000 rpm for 3 hours, and filtered through 0.2 μmcellulose acetate membranes, stimulate cell proliferation and DNAsynthesis in several human, canine, murine, and rat primary cultures andcell lines. In primary cultures of a dysgenic human neuroblastoma andembryonic brain of C57Bl fetal mice, the appearance of transformationfoci in periods of 20 to 30 days was detected, which evidencedchromosomal abnormalities. The same UCHT1-conditioned media HSSpreparation, when ultra-filtered in CENTRICON (AMICON) molecular filtermembranes, at cut levels of 10, 20, and 100 kD, the pro-proliferatingactivity was recovered in the 30–100 kD range. The fraction proved verystable (thermoresistant; resistant to trypsin). Precipitation of thesoluble, 0.2 μm filtered, UCHT1-conditioned media HSS preparation, withacetone 25%, 40%, 50%, 60%, and 80%, shows that the fraction of 40%retained the greatest proliferation inducing activity, using KGFR cellsgrowing in serum free conditions (FIGS. 25 and 32A–D). Differences withculture media and animal sera are present when using ionic exchangechromatography (FIG. 26), where UCHT1 media exhibits a larger peak atapproximately 11 ml, suggesting the presence of secreted protein.Anionic chromatography gives values of resolutions for pure albumin andtransferrin of 1.7, which is less than that seen for UCHT1 conditionedmedia (FIGS. 27 and 28), suggesting the presence of proteins other thanthese two. Further, ioselectric focusing gels indicate albuminpredominates as a protein in conditioned media, the attenuation in theextremities of the peak suggest the presence of other protein, but theresolution is insufficient to discriminate them (FIG. 29). Hydrophobicinteraction chromatography of conditioned media gives a pattern similarto the combination of transferrin and albumin, with slight differencesin correspondence, which could be due to non-specific proteininteractions (FIGS. 30 and 31).

FIG. 25 shows SDS-PAGE dyed with Coomassie blue stain at everysaturation level in precipitation with ammonium sulfate, as shown inTable 7. The first colunm contains molecular weight markers. FIG. 25shows that the main components are located at approximately 65 kD and 15kD, which are associated with albumin and lactoalbumin, respectively.Most protein precipitates at 65%–80% of saturation with ammoniumsulfate, but there are a greater number of proteins in the range of40%–50% and 50%–65%. Theoretically, thyroglobulin precipitates at40%–50%.

TABLE 6 Culture Line media % Fetal serum % Bovine Serum Other UCHT-1D/F12 2.5 10.0 — ″ ″ 2.0 — — ″ ″ 2.3 9.0 * ″ ″ 1.9 7.5 * ″ ″ 1.3 5.0 * ″″ 0.6 2.5 * ″ ″ 0.3 1.0 * KGFR D/F12 2.5 10.0 — Neuroblastoma D/F12 10.010.0 NGF ″ ″ 7.5 10.0 ″ NRK 52E D/F12 10.0 — — DMEM 10.0 — —

Table 6: Summary of cell lines cultured and the respective cultureconditions to which they have adapted. * Insulin, Transferrin,Somatostatin, Hydrocortisone, GlyLysElis, and TSH.

TABLE 7 Sample Absorbance Protein [μg/μl] 30% 3.336 38.688 37% 1.83311.656 45% 3.168 35.681 60% 3.039 33.387 80% 1.239 0.961 Blank 0.2010.000

Table 7: Results of quantitation of protein (BCA method) in differentacetone precipitations of UCHT1-CM (shown as %). Values are normalizedto the volume of the re-suspended pellet in 1 ml sterile PBS.

TABLE 8 Protein mg/ml CM 2% FS 0.997 HSS 10-2.5% 0.662 CM 10-2.5% 6.482CM 10-2.5% Pellet 30% 0.035 CM 10-2.5% N/S 30% 0.013 CM-N/S 80% 1.536CM-N/S 60% 4.231 CM-N/S 50% 8.195 CM-N/S 40% 0.108 CM-N/S 25% 3.411

Table 8: Results of protein quantitation by Bradford's method. CM:UCHT1-conditioned media, ES: fetal serum, 10–2.5%: complex media, N/S:no serum. Values of acetone precipitation are normalized to that of theresuspended pellet.

TABLE 9 Protein mg/ml Saturation 0%–20% 0.25 Saturation 20%–30% 2.11Saturation 30%–40% 5.17 Saturation 40%–50% 3.42 Saturation 50%–65% 3.73Saturation 65%–80% 11.91 Saturation 80%–95% 3.24

Table 9: Protein quantitation by Bradford's method, for desalinizationof ammonium sulfate precipitate. Results show a protein dilution 1:4 ofthe pellet.

EXAMPLE 17 Purification and Characterization of Tumor Cell LineProliferation Factors

The original supernatant of the tumor cell line culture, together withsubfractions thereof, are used to compare their effectiveness inimmortalizing some normal cell type. Preferably, the normal cell line isone that becomes rapidly immortalized. Next, the tumor supernatantfraction is size fractionated on gel filtration columns, preferablyusing HPLC technology. Next, which of the fractions the immortalizingcomponent resides in is determined. Once the proliferation-bearingfraction or fractions are identified, the active fractions are furthersubfractioned using HPLC, or general laboratory chromatography columnsthat separate proteins on the basis of charge, hydrophobicity, oradsorption, for example. Both one-dimensional and two-dimensionalSDS-PAGE gels are then run to assay the purity of the fractions. If thefractions are not pure, further separation can be conducted on the basisof size, charge, adsorption, etc., until the number of bands that appearon the gels is reduced to the smallest possible number. These fractionsare then cut out of the gels.

Once the fractions are purified, any among a battery of tests(particularly bioassays) of the purified fractions can be carried out toevaluate the effects of the fractions on eukaryotic or prokaryoticcells. For example, direct or indirect measurements of cellularproliferation can be utilized, which often involve incorporation of alabeled nucleoside into genomic DNA. Specific examples include tritiatedthymidine (³H-dT) and bromodeoxyoridine (BrdN) methods (Waldman et al.,Modern Pathol., 1991, 4:718–722; Gratzner, Science, 1982, 218:474–475;U.S. Pat. No. 6,461,806). Assays at the genetic level can also becarried out. For example, the Ames test, micronucleus test, comet assayon Tradescantia nuclei, or the pink mutation test on Tradescantiastaminal hairs, can be used. The Ames test (also referred to as thereverse mutation assay) is widely used to evaluate the mutagenicpotential of test substances, such as chemicals, formulations, orextracts (Ames, B. et al., Proc. Natl. Acad. Sci. USA, 1973, 70:782–786;McCann, J. et al., Proc. Natl. Acad. Sci. USA, 1975, 72:979–983; McCann,J. et al., Proc. Natl. Acad. Sci. USA, 1975, 72:5135–5139; Mortelmans,K. and E. Zeiger Mutat. Res., 2000, 455:29–60). The micronucleus test isused to screen test substances for clastogenic (chromosome-breaking) andaneugenic (loss of whole chromosome) activity. The test is based on theobservation that mitotic cells with chromosome breaks exhibitdisturbances in the anaphase distribution of their chromatin. After thetelophase, the displaced chromatin can be excluded from the nuclei ofthe daughter cells and is found in the cytoplasm as a micronuclei(Schmid, W., Mutation Res., 1975, 31:9; Salamone et al., Mutation Res.,1980, 74:347; Salamone, M. F., Mutation Res., 1983, 123:61; U.S. Pat.No. 6,387,618). Scoring of micronuclei can be performed relativelyeasily and on a variety of cell types, such as lymphocytes, fibroblasts,and exfoliated epithelial cells. As indicated above, tests forgenotoxicity using Tradescantia can also be used (Ichikawa, S., Mutat.Res., 1992, 270:3–22; Alvarez-Moya, C. et al., Salud Publica de México,November-December 2001, 43(6): 1–7). Other applicable assays aredisclosed in Ames, A. et al. “An Improved Bacterial Test System for theDetection and Classification of Mutagens and Carcinogens”, in Miller, J.ed. Discovering Molecular Genetics, Los Angeles: Cold Spring HarborLaboratory Press, 1996, pp. 367–376; U.S. Pat. No. 6,489,099; U.S. Pat.No. 6,461,806; U.S. Pat. No. 6,010,846; and U.S. Pat. No. 5,910,403).

Preferably, the cell type used in the bioassay(s) would be one that canbe immortalized by the proliferation factor in a rapid manner. The cellscan then be compared to the wild type starting material. Examples ofsuitable cell lines for use in the bioassays include, but are notlimited to, NIH-3T3, CHO, MDCK, KGFR, PTK2 (having only 14 chromosomes,this cell line is useful for determination of alterations at the genomiclevel), Indian Muntjac cells (which has six chromosomes), BALB/c-3T3(immortalized mouse cell line), C3H 10T1/2 (immortalized mouse cellline), RLV (virally infected rat cell line), and SA7 (virally infectedrat cell line. In addition, the Syrian Hamster Embryo CellTransformation Assay can be used (Kerckaert, G. et al., The Second NIEHSPredictive-Toxicology Evaluation Experiment: 30 Chemical CarcinogenicityBioassays; Environmental Health Perspectives 104, Supp. 5, [October,1996 ] “Use of the Syrian Hamster Embryo Cell Transformation Assay forCarcinogenicity Prediction of Chemicals Currently Being Tested by theNational Toxicology Program in Rodent Bioassays”). The cells can besuspended in a chemically defined medium without protein.Two-dimensional gels are then run against subfractions from both celltypes (starting material and treated material). Components that can beexamined include, but are not limited to, organelles such as thenucleus, endoplasmic reticulum, plasma membrane, and mitochondria.Fractionation would be performed with ion exchange, hydrophobic, and gelfiltration chromatography. Next, certain unique protein fractions fromthe proliferating cells would be identified. The fraction that appearsfirst would be preferred for the most rapid assay. A mass spectroscopyanalysis of the molecular weight of the fastest, uniquely expressedprotein (preferably to four significant figures), would identify whichprotein fragment is present. This protein can then be sequenced oridentified from a protein library. The rapid assay would then be toidentify the two-dimensional gel protein fraction that first correlateswith immortalization.

Once the protein is fully purified, the protein can be compared to agene library for determination of the nucleic acid sequence encoding theprotein. The gene can then be amplified. The cDNA and an operably linkedpromoter can be inserted into a plasmid for transfection into a suitablehost, such as bacteria, to recombinantly produce the protein inducingcell proliferation.

EXAMPLE 18 Target Cells

As described previously, there are over 200 cell types in the human bodyand the methods of the subject invention are useful in proliferating anyof these cell types, therapeutic, manufacturing, or other purposes.Examples of cell types that can be proliferated using methods of thesubject invention are listed in the table below. Other examples of celltypes that can be proliferated are disclosed herein.

TABLE 10 Examples of Target Cells Keratinizing Epithelial Cellskeratinocyte of epidermis basal cell of epidermis keratinocyte offingernails and toenails basal cell of nail bed hair shaft cellsmedullary cortical cuticular hair-root sheath cells cuticular ofHuxley's layer of Henle's layer external hair matrix cell Cells of WetStratified Barrier Epithelia surface epithelial cell of stratifiedsquamous epithelium of cornea tongue, oral cavity, esophagus, analcanal, distal urethra, vagina basal cell of these epithelia cell ofurinary epithelium Epithelial Cells Specialized for Exocrine Secretioncells of salivary gland mucous cell serous cell cell of von Ebner'sgland in tongue cell of mammary gland, secreting milk cell of lacrimalgland, secreting tears cell of ceruminous gland of ear, secreting waxcell of eccrine sweat gland, secreting glycoproteins cell of eccrinesweat gland, secreting small molecules cell of apocrine sweat gland cellof gland of Moll in eyelid cell of sebaceous gland, secreting lipid-richsebum cell of Bowman's gland in nose cell of Brunner's gland induodenum, secreting alkaline solution of mucus and enzymes cell ofseminal vesicle, secreting components of seminal fluid, includingfructose cell of prostate gland, secreting other components of seminalfluid cell of bulbourethral gland, secreting mucus cell of Bartholin'sgland, secreting vaginal lubricant cell of gland of Littré, secretingmucus cell of endometrium of uterus, secreting mainly carbohydratesisolated goblet cell of respiratory and digestive tracts, secretingmucus mucous cell of lining of stomach zymogenic cell of gastric gland,secreting pepsinogen oxyntic cell of gastric gland, secreting HCl acinarcell of pancreas, secreting digestive enzymes and bicarbonate Panethcell of small intestine, secreting lysozyme type II pneumocyte of lung,secreting surfactant Clara cell of lung Cells Specialized for Secretionof Hormones cells of anterior pituitary, secreting growth hormonefollicle-stimulating hormone luteinizing hormone prolactinadrenocorticotropic hormone thyroid-stimulating hormone cell ofintermediate pituitary, secreting melanocyte-stimulating hormone cellsof posterior pituitary, secreting oxytocin vasopressin cells of gut andrespiratory tract, secreting serotonin endorphin somatostatin gastrinsecretin cholecystokinin insulin glucagons bombesin cells of thyroidgland, secreting thyroid hormone calcitonin cells of parathyroid gland,secreting parathyroid hormone oxyphil cell cells of adrenal gland,secreting epinephrine norepinephrine steroid hormones mineralocorticoidsglucocorticoids cells of gonads, secreting testosterone estrogenprogesterone cells of juxtaglomerular apparatus of kidneyjuxtaglomerular cell macula densa cell peripolar cell mesangial cellEpithelial Absorptive Cells in Gut, Exocrine Glands, and UrogenitalTract brush border cell of intestine striated duct cell of exocrineglands gall bladder epithelial cell brush border cell of proximal tubuleof kidney distal tubule cell of kidney nonciliated cell of ductulusefferens epididymal principal cell epididymal basal cell CellsSpecialized for Metabolism and Storage hepatocyte fat cells (e.g.,adipocyte) white fat brown fat lipocyte of liver Epithelial CellsServing Primarily a Barrier Function, Lining the Lung, Gut, ExocrineGlands, and Urogenital Tract type I pneumocyte pancreatic duct cellnonstriated duct cell of sweat gland, salivary gland, mammary gland,etc. parietal cell of kidney glomerulus podocyte of kidney glomeruluscell of thin segment of loop of Henle collecting duct cell duct cell ofseminal vesicle, prostate gland, etc. Epithelial Cells Lining ClosedInternal Body Cavities vascular endothelial cells of blood vessels andlymphatics (e.g., microvascular cell) fenestrated continuous splenicsynovial cell serosal cell squamous cell lining perilymphatic space ofear cells lining endolymphatic space of ear squamous cell columnar cellsof endolymphatic sac with microvilli without microvilli “dark” cellvestibular membrane cell stria vascularis basal cell stria vascularismarginal cell cell of Claudius cell of Boettcher choroid plexus cellsquamous cell of pia-arachnoid cells of ciliary epithelium of eyepigmented nonpigmented corneal “endothelial” cell Ciliated Cells withPropulsive Function of respiratory tract of oviduct and of endometriumof uterus of rete testis and ductulus efferens of central nervous systemCells Specialized for Secretion of Extracellular Matrix epithelial:ameloblast planum semilunatum cell of vestibular apparatus of earinterdental cell of organ of Corti nonepithelial: fibroblasts pericyteof blood capillary (Rouget cell) nucleus pulposus cell of intervertebraldisc cementoblast/cementocyte odontoblast/odontocyte chondrocytes ofhyaline cartilage of fibrocartilage of elastic cartilageosteoblast/osteocyte osteoprogenitor cell hyalocyte of vitreous body ofeye stellate cell of perilymphatic space of ear Contractile Cellsskeletal muscle cells red white intermediate muscle spindle-nuclear bagmuscle spindle-nuclear chain satellite cell heart muscle cells ordinarynodal Purkinje fiber Cardiac valve tissue smooth muscle cellsmyoepithelial cells: of iris of exocrine glands Cells of Blood andImmune System red blood cell (erythrocyte) megakaryocyte macrophagesmonocyte connective tissue macrophage Langerhan's cell osteoclastdendritic cell microglial cell neutrophil eosinophil basophil mast cellplasma cell T lymphocyte helper T cell suppressor T cell killer T cell Blymphocyte IgM IgG IgA IgE killer cell stem cells and committedprogenitors for the blood and immune system Sensory Transducersphotoreceptors rod cones blue sensitive green sensitive red sensitivehearing inner hair cell of organ of Corti outer hair cell of organ ofCorti acceleration and gravity type I hair cell of vestibular apparatusof ear type II hair cell of vestibular apparatus of ear taste type IItaste bud cell smell olfactory neuron basal cell of olfactory epitheliumblood pH carotid body cell type I type II touch Merkel cell of epidermisprimary sensory neurons specialized for touch temperature primarysensory neurons specialized for temperature cold sensitive heatsensitive pain primary sensory neurons specialized for painconfigurations and forces in musculoskeletal system proprioceptiveprimary sensory neurons Autonomic Neurons cholinergic adrenergicpeptidergic Supporting Cells of Sense Organs and of Peripheral Neuronssupporting cells of organ of Corti inner pillar cell outer pillar cellinner phalangeal cell outer phalangeal cell border cell Hensen cellsupporting cell of vestibular apparatus supporting cell of taste budsupporting cell of olfactory epithelium Schwann cell satellite cellenteric glial cell Neurons and Glial Cells of Central Nervous Systemneurons glial cells astrocyte oligodendrocyte Lens Cells anterior lensepithelial cell lens fiber Pigment Cells melanocyte retinal pigmentedepithelial cell iris pigment epithelial cell Germ Cells oogonium/oocytespermatocyte Spermatogonium blast cells fertilized ovum Nurse Cellsovarian follicle cell Sertoli cell thymus epithelial cell (e.g.,reticular cell) placental cell

1. A UCHT1 rat thyroid cell line, having deposit number DSM ACC2535. 2.A method for proliferating cells, comprising culturing one or moretarget cells with conditioned medium prepared from the UCHT1 rat thyroidcell line having deposit No. DSM AC2535 for a period of time sufficientto increase the proliferation potential of the one or more target cells.3. A method for producing a continuous cell line, comprising culturingone or more target cells with conditioned medium prepared from the UCHT1rat thyroid cell line having deposit No. DSM ACC2535 for a period oftime sufficient to increase the proliferation potential of the one ormore target cells such that the one or more target cells proliferateindefinitely.
 4. The method of claim 3, wherein the period of time iswithin the range of about 1 month to about 8 months.
 5. The method ofclaim 3, wherein the one or more target cells are mammalian cells. 6.The method of claim 3, wherein the one or more target cells are humancells.
 7. The method of claim 3, wherein the one or more target cellsproduce a biomolecule, and wherein said method further comprisesharvesting the biomolecule from the one or more target cells followingsaid culturing.
 8. The method of claim 3, wherein the one or more targetcells are selected from the group consisting of skeletal muscle cells,neural cells, myocardial cells, hypothalamus cells, atrial cardiocytes,corneal endothelial cells, and pancreatic cells.
 9. A method fordetermining the effect of an agent on one or more cells, comprisingculturing one or more cells with conditioned medium prepared from theUCHT1 rat thyroid cell line having deposit No. DSM ACC2535 for a periodof time sufficient to increase the proliferation potential of the one ormore cells, exposing the one or more cells to an agent to be tested, anddetermining the effect of the agent on the one or more cells.
 10. Themethod of claim 9, wherein said exposing comprises contacting the one ormore cells to the agent to be tested.